Methods for isolating molecular mimetics of unique Neisseria meningitidis serogroups B epitopes

ABSTRACT

Novel bactericidal antibodies against  Neisseria meningitidis  serogroup B (“MenB”) are disclosed. The antibodies either do not cross-react or minimally cross-react with host tissue polysialic acid and hence pose minimal risk of autoimmune activity. The antibodies are used to identify molecular mimetics of unique epitopes found on MenB or  E. coli  Kl. Examples of such peptide mimetics are described that elicit serum antibody capable of activating complement-mediated bacteriolysis of MenB. Vaccine compositions containing such mimetics can be used to prevent MenB or  E. coli  Kl disease without the risk of evoking autoantibody.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is related to provisional patent applicationserial No. 60/025,799, filed Aug. 27, 1996, from which priority isclaimed under 35 U.S.C. §119(e)(1) and which is incorporated herein byreference in its entirety.

TECHNICAL FIELD

[0002] The present invention pertains generally to bacterial pathogens.In particular, the invention relates to antibodies that elicitfunctional activity against Neisseria meningitidis serogroup B and alsolack autoimmune activity, methods of obtaining and using the same, aswell as molecular mimetics identified using the antibodies.

BACKGROUND OF THE INVENTION

[0003]Neisseria meningitidis is a causative agent of bacterialmeningitis and sepsis. Meningococci are divided into serological groupsbased on the immunological characteristics of capsular and cell wallantigens. Currently recognized serogroups include A, B, C, D, W-135, X,Y, Z and 29E. The polysaccharides responsible for the serogroupspecificity have been purified from several of these groups, includingA, B, C, D, W-135 and Y.

[0004]N. meningitidis serogroup B (“MenB”) accounts for approximately 50percent of bacterial meningitis in infants and children residing in theU.S. and Europe. The organism also causes fatal sepsis in young adults.In adolescents, experimental MenB vaccines consisting of outer membraneprotein (OMP) vesicles have been found to be approximately 50%protective. However, no protection has been observed in vaccinatedinfants and children, the age groups at greatest risk of disease.Additionally, OMP vaccines are serotype- and subtype-specific, and thedominant MenB strains are subject to both geographic and temporalvariation, limiting the usefulness of such vaccines.

[0005] Effective capsular polysaccharide-based vaccines have beendeveloped against meningococcal disease caused by serogroups A, C, Y andW135. However, similar attempts to develop a MenB polysaccharide vaccinehave failed due to the poor immunogenicity of the capsular MenBpolysaccharide (termed “MenB PS” herein). MenB PS is a homopolymer of(N-acetyl (a2e8) neuraminic acid. Escherichia coli Kl has the identicalcapsular polysaccharide. Antibodies elicited by MenB PS cross-react withhost polysialic acid (PSA). PSA is abundantly expressed in fetal andnewborn tissue, especially on neural cell adhesion molecules (“NCAMs”)found in brain tissue. PSA is also found to a lesser extent in adulttissues including in kidney, heart and the olfactory nerve. Thus, mostanti-MenB PS antibodies are also autoantibodies. Such antibodiestherefore have the potential to adversely affect fetal development, orto lead to autoimmune disease.

[0006] MenB PS derivatives have been prepared in an attempt tocircumvent the poor immunogenicity of MenB PS. For example, C₃-C₈N-acyl-substituted MenB PS derivatives have been described. See, EPPublication No. 504,202 B, to Jennings et al. Similarly, U.S. Pat. No.4,727,136 to Jennings et al. describes an N-propionylated MenB PSmolecule, termed “NPr-MenB PS” herein. Mice immunized with NPr-MenB PSglycoconjugates were reported to elicit high titers of IgG antibodies.Jennings et al. (1986) J. Immunol. 137:1708. In rabbits, two distinctpopulations of antibodies, purportedly associated with two differentepitopes, one shared by native MenB PS and one unshared, were producedusing the derivative. Bactericidal activity was found in the antibodypopulation that did not cross react with MenB PS. Jennings et al. (1987)J. Exp. Med. 155:1207. The identity of the bacterial surface epitope(s)reacting with the protective antibodies elicited by this conjugateremains unknown.

[0007] Peptides can serve as mimics of polysaccharides by binding topolysaccharide-specific antibodies as well as to other polysaccharidebinding proteins. For example, concanavalin A (Con A), which binds tooligosaccharides bearing terminal alpha-linked mannose or glucoseresidues, has been used to select peptide mimetics from random librariesof bacterial phage bearing short peptide sequences at the amino-terminusof the pIII coat protein. Olderberg et al. (1992) Proc. Natl. Acad. Sci.USA 89:5393, Scott et al. (1992) Proc. Natl. Acad. Sci. USA 89:5398.Similarly, monoclonal antibodies have identified peptide mimetics of acarbohydrate present on the surface of adinocarcinona cells from a phagelibrary Hoess at al. (1993) Gene 120:43.

[0008] Peptides can also elicit polysaccharide-specific antibodies. Forexample, Westerink et al. (1988) Infect. Immun. 56:1120, used amonoclonal antibody to the N. meningitidis serogroup C (“MenC”) capsularploysacceride to elicit an anti-idiotype antibody. Mice immuniced withthe anti-idiotype antibody were protected against infection with alethal dose of MenC bacteria. These experimenters subsequentlydemonstrated that a peptide fragment of a MenC anti-idiotype antibodyeliciten serum anti-MenC antibodies and protected animals frombacteremia and death after lethal challenge with MenC bacteria.Wescerink et al. (1995) Proc. Natl. Acad. Sci USA 92:4021.

[0009] However, to date, no such approach has been taken with respect toMenB vaccine development. It is readily apparent that the production ofa safe and effective vaccine against MenB would be particularlydesirable.

SUMMARY OF THE INVENTION

[0010] The present invention is based on the discovery of functionallyactive antibodies directed against MenB PS derivatives, wherein theantibodies do not cross-react, on are minimally cross-reactive, withhost tissues as determined using the assays described herein. Theseantibodies therefore pose minimal risk of evoking autoimmune disease andare termed “non-autoreactive” herein. Assays used herein to determineautoreactivity include binding assays against a neuroblastoma cell lineexpressing long chain polysiatic acid residues on the cell surface.Specifically, antibodies that are negative in these assays areconsidered to lack autoreactivity. The non-autoreactive antibodies areparticularly useful for identifyied molecular mimetics of unique MenB PSepitopes that can be used in vaccine compositions. Furthermore, theantibodies humanized versions of the antibodies fragments and functionalequivalents thereof, will also and use in passive immunization against,and/or to a adjunct to therapy for, MenB and E. coli Kl disease Sincesuch molecules do not bind to polysialic acid in tissues as determinedby the autoreactivity assays described herein, they provide a safe andefficacious method for the treatment and/or prevention of MenB and E.coli Kl disease.

[0011] Accordingly, in one embodiment, the subject invention relates toantibodies directed against MenB PS derivatives, wherein the antibodiesare not autoreactive with host tissue. Such antibodies may further becharacterized as being capable of eliciting functional activity againstMenB bacteria. One particular group of such antibodies is alsocharacterized as non cross-reactive with Neisseria meningitidisserogroup B capsular polysaccharide (NAc-MenB PS) in an ELISA. However,these antibodies are anti-capsular in that they can bind to the cellsurface of a Group B encapsulated bacteria, but not tocapsular-deficient mutants.

[0012] Another embodiment of the invention relates to monoclonalantibodies directed against MenB PS derivatives, and hybridomasproducing those monoclonal antibodies.

[0013] Other embodiments of the invention relate to unique Neisseriameningitidis serogroup B epitopes that are capable of being bound by theantibody molecules of the present invention.

[0014] Still further embodiments of the subject invention are related tomethods for isolating molecular mimetics of unique epitopes of MenB PSand molecular mimetics identified using the methods. The methodscomprise:

[0015] (a) providing a population of molecules including a putativemolecular mimetic of a unique epitope of MenB PS;

[0016] (b) contacting the population of molecules with the antibodiesdescribed above under conditions that allow immunological bindingbetween the antibody and the molecular mimetic, if present, to provide acomplex; and

[0017] (c) separating the complexes from non-bound molecules.

[0018] In another embodiment, the subject invention is directed to avaccine composition comprising a unique epitope of MenB in combinationwith a pharmaceutically acceptable excipient.

[0019] In yet another embodiment, the invention is directed to a vaccinecomposition comprising a molecular mimetic of a unique epitope of MenBin combination with a pharmaceutically acceptable excipient.

[0020] In still a further embodiment, the invention is directed to avaccine composition comprising an anti-idiotypic antibody molecularmimetic of a unique epitope of MenB in combination with apharmaceutically acceptable excipient.

[0021] In yet further embodiments, the invention relates topharmaceutical compositions comprising the antibodies described above.

[0022] In another embodiment, the subject invention is directed to amethod for treating or preventing MenB and/or E. coli Kl disease in amammalian subject comprising administering an effective amount of theabove pharmaceutical compositions to the subject.

[0023] These and other embodiments of the present invention will readilyoccur to those of ordinary skill in the art in view of the disclosureherein.

BRIEF DESCRIPTION OF THE FIGURES

[0024] FIGS. 1A-1D depict dose-response binding activity of threerepresentative anti-NPr-MenB PS monoclonal antibodies (SEAM-3, SEAM-5,SEAM-16 and SEAM-18, respectively), to solid phase NPr-MenB PS asdetermined by ELISA. Data shown are for the antibodies diluted in buffer( ), or in buffer containing 25 μg/ml of soluble NPr-MenB PS (∘).Different ranges for the X axis in the data are used, wherein monoclonalantibodies SEAM-3, SEAM-16, and SEAM-18 are shown at 0.0001 to 1 μg/ml,and monoclonal antibody SEAM-5 is shown at 0.1 to 100 μg/ml.

[0025]FIG. 2 depicts the inhibition of binding of four representativeanti-NPr-MenB PS monoclonal antibodies (SEAM-2, SEAM-3, SEAM-16 andSEAM-18) to solid phase NPr-MenB PS by either 25 μg/ml of soluble highmolecular weight (HMW) NPr-MenB PS inhibitor (▪), or 25 μg/ml of lowmolecular weight (LMW) NPr-MenB oligosaccharide inhibitor having anaverage degree of polymerization of 3.8 monomers (□), as determined byELISA.

[0026]FIG. 3 depicts the binding of five representative anti-NPr-MenB PSmonoclonal antibodies (SEAM-12, SEAM-16, SEAM-18, SEAM-2, and SEAM-3) tosolid phase NAc-MenB PS as determined by ELISA. Three of the antibodies,SEAM-12, SEAM-16 and SEAM-18, showed significant binding when tested at0.5 and/or 5 μg/ml of antibody. Two other antibodies, SEAM-2 and SEAM-3,were negative when tested at 5-fold higher concentrations (25 μg/ml ofantibody).

[0027] FIGS. 4A-4G depict the cross-reactivity of control antibodies andrepresentative anti-NPr-MenB PS monoclonal antibodies (SEAM-3, SEAM-18,SEAM-9, SEAM-10, and SEAM-7) with encapsulated and non-encapsulatedwhole MenB bacteria as determined by indirect fluorescence flowcytometry. The capsule contains NAc-MenB PS.

[0028] FIGS. 5A-5D depict the complement-mediated bactericidal activityof four representative anti-NPr-MenB PS monoclonal antibodies (SEAM-3,SEAM-5, SEAM-12, and SEAM-18, respectively) when tested against the MenBtest strain 8047. Results are shown from experiments with threedifferent complement sources: infant rabbit complement I (▴); infantrabbit complement II ( ); and human complement (∘).

[0029] FIGS. 6A-6I depict the cross-reactivity of three controlantibodies and four representative anti-NPr-MenB PS monoclonalantibodies (SEAM-5, SEAM-35, SEAM-12, and SEAM-7) with polysialic acidantigens displayed on the surface of the human neuroblastoma cell lineCHP-134 as determined by indirect fluorescence flow cytometry.

[0030]FIG. 7 depicts the amino acid sequences of 67 unique peptidemimetic sequences (SEQ ID NOs. 1-67) selected by SEAM monoclonalantibodies from phage display peptide libraries.

[0031]FIGS. 8A and 8B depict the ELISA binding activity of sevenrepresentative SEAM monoclonal antibodies (SEAM-2, SEAM-3, SEAM-5,SEAM-7, SEAM-12, SEAM-16, and SEAM-18) to two peptides containingpeptide mimetic sequences selected by SEAM monoclonal antibodies. (InFIG. 8A, “Pep 4” is Lauryl-GLY-GLY-[SEQ ID NO. 4]-Amide, and in FIG. 8B,“Pep 8” is Lauryl-GLY-GLY-[SEQ ID NO. 8]-Amide). Each peptide contains acarboxyl terminal amide and a Lauryl-Gly-Gly at the amino terminal endin order to facilitate binding of the peptide to the microtiter plate.

[0032]FIG. 9 depicts the antibody binding activity of pooled (four miceper pool) immune and unimmunized (CTL) sera from CD1 mice as measured byan ELISA with peptide Pep 8 as the solid phase antigen. The immune serawere from mice immunized with 5 μg or 50 μg of mimetic peptidescomplexed to the capsule-deficient Neisseria meningitidis Strain M7outer membrane protein vesicles. The peptides included Pep 5(Lauryl-GLY-GLY-[SEQ ID NO. 5]-Amide), Pep 8 (Lauryl-GLY-GLY-[SEQ ID NO.8]-Amide), or a mixture of nine peptides Pep 1 through Pep 9 (Pep 1,Lauryl-GLY-GLY-[SEQ ID NO. 1]-Amide; Pep 2, Lauryl-GLY-GLY-[SEQ ID NO.2]-Amide; Pep 3, Lauryl-GLY-GLY-[SEQ ID NO. 31-Amide; Pep 4,Lauryl-GLY-GLY-[SEQ ID NO. 4]-Amide; Pep 5, Lauryl-GLY-GLY-[SEQ ID NO.5]-Amide; Pep 6, Lauryl-GLY-GLY-[SEQ ID NO. 6]-Amide; Pep 7,Lauryl-GLY-GLY-[SEQ ID NO. 7]-Amide; Pep 8, Lauryl-GLY-GLY-[SEQ ID NO.8]-Amide; and Pep 9, Lauryl-GLY-GLY-[SEQ ID NO. 9)-Amide). Binding iscompared between sera diluted in buffer (□), buffer containing solublePep 8 (Acetyl-[SE ID NO. 8]-Amide) (▪), or buffer containing a solubleirrelevant peptide R1 (Acetyl-GLN-TRP-GLU-ARG-THR-TYR-Amide (SEQ ID NO.68)) (cross-hatched bars).

[0033]FIG. 10 depicts the antibody binding activity of pooled (four miceper pool) immune and unimmunized control sera from CD1 mice as measuredby an ELISA with NPr-MenB PS as the solid phase antigen. The mice wereimmunized with the peptide immunogens as described above in FIG. 9.

[0034]FIG. 11 depicts the antibody binding activity of pooled (four miceper pool) immune and unimmunized control sera from CD1 mice as measuredby an ELISA with NAc-MenB PS as the solid phase antigen. The mice wereimmunized with the peptide immunogens as described above in FIG. 9. TheSEAM-30 antibody, with known autoantibody activity, served as thepositive control.

[0035] FIGS. 12A-12B depict the percent survival of bacteria incubatedwith various dilutions of test sera and human complement. The data shownare from testing pooled sera (four mice per pool) from CD1 miceimmunized with 5 μg (FIG. 11A) or 50 μg (FIG. 11B) of mimetic peptidePep 8 (Lauryl-GLY-GLY-[SEQ ID NO. 8]-Amide) complexed tocapsular-deficient Neisseria meningitidis Strain M7 outer membraneprotein vesicles. The sera were diluted in buffer, or in buffercontaining Pep 8 inhibitor (100 μg/ml). The source of complement washuman agammaglobulinemia and the bacterial test strain was 8047.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The practice of the present invention will employ, unlessotherwise indicated, conventional methods of immunology, microbiology,molecular biology and recombinant DNA techniques within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition,1989); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weirand C. C. Blackwell eds., 1986, Blackwell Scientific Publications).

[0037] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0038] As used in this specification and the appended claims, thesingular forms “a,” “an” and “the” include plural references unless thecontent clearly dictates otherwise.

I. Definitions

[0039] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0040] As used herein, a “MenB PS derivative” refers to a moleculeobtained by the chemical modification of the native capsularpolysaccharide of MenB. Such MenB PS derivatives include, but are notlimited to, MenB PS molecules which have been modified by thesubstitution of sialic acid residue N-acetyl groups of the nativemolecule with appropriate acyl groups, such as C₃-C₈, and higher, acylgroups wherein the term “acyl group” encompasses any acylated linear,branched, aliphatic or aromatic molecule. A particularly preferred MenBPS derivative for use herein comprises the substitution of N-propionylgroups for N-acetyl groups of native MenB PS (termed “NPr-MenB PS”herein). Methods for synthesizing N-acyl-substituted MenB PSderivatives, including NPr-MenB PS, are known in the art and describedin e.g., U.S. Pat. No. 4,727,136 to Jennings et al. and EP PublicationNo. 504,202 B, also to Jennings et al. “Molecular mimetics” of MenB PS,or derivatives of MenB PS are molecules that functionally mimic at leastone “unique” epitope expressed on a MenB bacteria. A “unique epitope” isan epitope capable of eliciting the formation of functionally active(e.g., opsonic and/or complement-mediated bactericidal) anti-MenBantibodies that either are not cross-reactive with polysialic acid inhost tissue and hence lack autoimmune activity, or are minimallycross-reactive. Such molecular mimetics are useful in vaccinecompositions and in eliciting antibodies for diagnostic or therapeuticapplications, as described further below. Molecular mimetics include,but are not limited to, small organic compounds; nucleic acids andnucleic acid derivatives; saccharides or oligosaccharides; peptidemimetics including peptides, proteins, and derivatives thereof, such aspeptides containing non-peptide organic moieties, synthetic peptideswhich may or may not contain amino acids and/or peptide bonds, butretain the structural and functional features of a peptide ligand, andpeptoids and oligopeptoids which are molecules comprising N-substitutedglycine, such as those described by Simon et al. (1992) Proc. Natl.Acad. Sci. USA 89:9367; and antibodies, including anti-idiotypeantibodies. Methods for the identification and production of molecularmimetics are described more fully below.

[0041] The term “antibody” encompasses polyclonal and monoclonalantibody preparations, as well as preparations including hybridantibodies, altered antibodies, F(ab′)₂ fragments, F(ab) molecules, Fvfragments, single domain antibodies, chimeric antibodies and functionalfragments thereof which exhibit immunological binding properties of theparent antibody molecule.

[0042] As used herein, the term “monoclonal antibody” refers to anantibody composition having a homogeneous antibody population. The termis not limited by the manner in which it is made. The term encompasseswhole immunoglobulin molecules, as well as Fab molecules, F(ab′)₂fragments, Fv fragments, and other molecules that exhibit immunologicalbinding properties of the parent monoclonal antibody molecule. Methodsof making polyclonal and monoclonal antibodies are known in the art anddescribed more fully below.

[0043] An “antigen” is defined herein to include any substance that maybe specifically bound by an antibody molecule. An “immunogen” is anantigen that is capable of initiating lymphocyte activation resulting inan antigen-specific immune response.

[0044] By “epitope” is meant a site on an antigen to which specific Bcells and T cells respond. The term is also used interchangeably with“antigenic determinant” or “antigenic determinant site.” A peptideepitope can comprise 3 or more amino acids in a spatial conformationunique to the epitope. Generally, an epitope consists of at least 5 suchamino acids and, more usually, consists of at least 8-10 such aminoacids. Methods of determining spatial conformation of amino acids areknown in the art and include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance spectroscopy. Furthermore, theidentification of epitopes in a given protein is readily accomplishedusing techniques well known in the art. See, e.g., Geysen et al. (1984)Proc. Natl. Acad. Sci. USA 81:3998 (general method of rapidlysynthesizing peptides to determine the location of immunogenic epitopesin a given antigen); U.S. Pat. No. 4,708,871 (procedures for identifyingand chemically synthesizing epitopes of antigens); and Geysen et al.(1986) Molecular Immunology 23:709 (technique for identifying peptideswith high affinity for a given antibody). Antibodies that recognize thesame epitope can be identified in a simple immunoassay showing theability of one antibody to block the binding of another antibody to atarget antigen.

[0045] A “unique MenB epitope” is defined herein as an epitope presenton a MenB bacterium, wherein antibodies directed toward the epitope arecapable of binding specifically to MenB and not cross reacting, orminimally cross reacting, with sialic acid residues present on thesurface of host tissue. Immunogens containing or mimicking one or more“unique MenB epitopes” are thus useful in vaccines for prevention ofMenB disease, and will not elicit an autoimmune response, or poseminimal risk of eliciting an autoimmune response.

[0046] An antibody displays “functional activity” against a MenBorganism when the antibody molecule exhibits complement-mediatedbactericidal activity and/or opsonic activity against MenB as determinedusing the assays described herein.

[0047] An antibody specific for a “unique” MenB epitope “lacksautoimmune activity,” and/or is “not autoreactive” when the subjectantibody does not exhibit cross-reactive immunological bindingproperties with polysialic acid in host tissue as determined using thebinding assays described herein.

[0048] An antibody specific for a “unique” MenB epitope is “notautoreactive” when the subject antibody requires approximately ten timesgreater antibody concentration to exhibit binding to polysialic acid inhost tissues, compared to a known cross-reactive auto antibodyconsidered positive in the binding assays described herein. (Forexample, compare binding of SEAM-12 to binding of SEAM-35 in FIG. 6).Thus, the term encompasses those antibodies that are not autoreactive orminimally autoreactive in the binding assays described herein.

[0049] As used herein, the terms “immunological binding,” and“immunological binding properties” refer to non-covalent interactions ofthe type which occur between an immunoglobulin molecule and an antigenfor which the immunoglobulin is specific.

[0050] By “purified” and “isolated” is meant, when referring to apolypeptide, antibody or nucleotide sequence, that the indicatedmolecule is present in the substantial absence of other biologicalmacromolecules of the same type. The terms “purified” and “isolated” asused herein preferably mean at least 75% by weight, more preferably atleast 85% by weight, more preferably still at least 95% by weight, andmost preferably at least 98% by weight, of biological macromolecules ofthe same type are present. Similarly, an “isolated” antibody is anantibody separated from a mixed population of antibodies, such as fromantisera raised against a molecule of interest.

[0051] “Homology” refers to the percent of identity between twopolynucleotide or polypeptide moieties. The correspondence between twoor more sequences can be determined by techniques known in the art. Forexample, homology can be determined by a direct comparison of thesequence information between two polypeptide molecules. Two peptidesequences are “substantially homologous” when at least about 60%(preferably at least about 80%, and most preferably at least about 90%)of the amino acids match.

II. Modes of Carrying Out the Invention

[0052] The present invention is based on the discovery of novelfunctional antibodies directed against MenB. The antibodies do notcross-react, or are minimally cross-reactive with polysialic acid inhost tissue as determined using the assays described herein, and hencethe antibodies have a lower risk of evoking autoimmune activity thanantibodies that are highly cross-reactive with host tissue. Theantibodies can be used to identify molecular mimetics of unique epitopesfound on the surface of MenB. The antibodies and/or mimetics can be usedin vaccine compositions to treat and/or prevent MenB and E. coli Kldisease, as well as in diagnostic compositions for the identification ofMenB and E. coli Kl bacteria.

[0053] As explained above, the native capsular polysaccharide of MenB,termed “MenB PS” herein, is poorly immunogenic in humans and othermammalian subjects. Furthermore, native MenB PS can elicit theproduction of autoantibodies and, hence, may be inappropriate for use invaccine compositions. Thus, the present invention uses antibodiesprepared against MenB PS derivatives. These antibodies are selectedbased on their ability to exhibit functional activity against MenBbacteria, wherein the functional activity is important in conferringprotection against MenB disease. The antibodies are also selected on thebasis of showing minimal or undetectable autoimmune activity.

[0054] More particularly, MenB PS derivatives were prepared for use inobtaining the antibody molecules of the present invention. Thederivatives generally comprise C₃-C₈ acyl substitutions of sialic acidresidue N-acetyl groups of the native molecule. Particularly preferredMenB PS derivatives comprise the substitution of N-propionyl groups forN-acetyl groups of native MenB PS and are termed “NPr-MenB PS” herein.Such derivatives and methods for synthesizing the same are described ine.g., U.S. Pat. No. 4,727,136 and EP Publication No. 504,202 B, both toJennings et al.

[0055] The C₃-C₈ acyl derivatives can be made by first treating nativeMenB (obtained from e.g., N. meningitidis cultures) in the presence of astrong base to quantitatively remove the N-acetyl groups and to providea reactive amine group in the sialic acid residue parts of the molecule.The deacylated MenB PS fragments are then N-acylated. For example, inthe case of NPr-MenB PS, the deacylated molecule is N-propionylatedusing a source of propionyl groups such as propionic anhydride orpropionyl chloride, as described in U.S. Pat. No. 4,727,136 to Jenningset al. The extent of N-acylation can be determined using, for example,NMR spectroscopy. In general, reaction conditions are selected such thatthe extent of N-acylation is at least about 80%.

[0056] In order to increase the immunogenicity of the MenB PSderivatives, the derivatives can be conjugated to a suitable carriermolecule to provide glycoconjugates. Particularly, N-acylated MenB PSglycoconjugate preparations having well defined and controlledstructural configurations can be formed from intermediate sizedN-acylated MenB oligosaccharides as described below.

[0057] Thus, a group of N-acylated MenB PS glycoconjugates, an exampleof which is termed “COMJ-2” herein can be prepared as follows. AnN-acylated MenB PS preparation, having substantially 100% N-acylatedMenB sialic acid residues, as determined by e.g., NMR analysis, can befragmented under mild acidic conditions to provide a population ofoligosaccharide molecules of varying sizes. The fragmented products aresize fractionated using for example standard ion exchangechromatographic techniques combined with e.g., stepwise salt gradients,to provde fractions of N-acylated MenB molecules of homogenous sizes.Fractions containing intermediate sized oligosaccharides e.g., with anaverage Dp or about 5 to about 22, preferably 10 to about 20, and moreparticularly about 12 to about 18, are chemically end-activated at thenon reducing termini and conjugated to protein carriers by a teductiveamination technique to provide the CONJ-2 glyoconjugation. Successfulconjugation can be determined by, e.g., gel filtration, and the finalsacchisde to protein ratio (w/w) assessed by colorimetric assay.

[0058] Glycoconjugated formed from MenB PS derivatives, such as theCONJ-2 are then used herein to elicit the formation of anti-saccharideantibodies in an immunized host a subset of such antibodies should bindto MenB bacteria should not cross-react, or be minimally cross-reactivewith host tissue sialic acid residues as determined the binding assaysdescribed herein. The antibodies can be fully characterized with respectto isotype the antigenic specificity, functional activity and crossreactivity with host tissue.

[0059] For example, mammalian subjects, conveniently. standardlaboratory animals such as rodents and rabbits, can be summarized withcompositions containing the glycocrjugates along with a suitableadjuvant to elicit the production of polyclonal sera. Groups of animalsare generally immunized and boosted several times with the compositions.Antisera from immunized animals can be obtained, and polyclonal serathat does not cross-react with host tissue can be obtained using in-situabsorption or conventional affinity chromatography techniques.Successful glycoconjugate antigens can be identified by their ability toelict a substantial IgG anti MenB PS derivative antibody response,charasteristic of a T-cell dependent antigen. Conjugates that are foundto be highly immunogenic and produce predominantly IgG antibodies areparticularly preferred for use in the methods of the present invention.

[0060] MenB PS derivatives that are capable of eliciting the formationof bactericidal antisera are suitable for use in the production ofmonoclonal antibodies. More particularly, the process used to providethe various MenB PS derivative conjugates is designed to producesuperior immunogens presenting unique saccharide-associated epitopesthat mimic those found on the surface of MenB organisms and areexpressed minimally in the host. The MenB PS derivatives describedherein and thus capable of eliciting the production of MenB-specificantibodies which can be use directly in the selective or therapeuticpharmaceutical preparation or, preferably, used to search for mimeticsof MenB polysaccharide antigens that will provide unique epitopes foranti-MenB vaccines.

[0061] Thus is one embodiment of the invention, selected MenBderivatives are used to provide monoclonal antibodies and functionalequivalents thereof. The term “functional equivalent” with respect to aparticular that: (a) cross-blocks an exemplified monoclonal antibodieskinds selectively to the MenB PS derivative or glycoconjugate inquestion; (c) does not cross-react, or minimally cross-reacts, with hostPSA as determined using the binding assays described herein; and,optionally, activity (e.g., complement-mediated bactericidal and/oropsonic activity) against MenB bacterial cells as determined by standardassays described below. Further, as used herein with regard to aparticular monoclonal antibody producing hybridoma of the invention, theterm “progeny” is intended to include all derivatives, issue, andoffspring of the parent hybridoma that produce the monoclonal antibodyproduced by the parent, regardless of generation or karyotypic identity.

[0062] Monoclonal antibodies are prepared using standard techniques,well known in the art, such as by the method of Kohler and Milstein,Nature (1975) 256:495, or a modification thereof, such as described byBuck et al. (1982) In Vitro 18:377. Typically, a mouse or rat isimmunized with the MenB PS derivative conjugated to a protein carrier,boosted and the spleen (and optionally several large lymph nodes)removed and dissociated into single cells. If desired, the spleen cellsmay be screened (after removal of non-specifically adherent cells) byapplying a cell suspension to a plate or well coated with the antigen.B-cells, expressing membrane-bound immunoglobulin specific for theantigen, will bind to the plate, and will not be rinsed away with therest of the suspension. Resulting B-cells, or all dissociated spleencells, are then induced to fuse with myeloma cells to form hybridomas.Representative murine myeloma lines for use in the hybridizationsinclude those available from the American Type Culture Collection(ATCC).

[0063] More particularly, somatic cell hybrids can be prepared by themethod of Buck et al., (supra), using the azaguanine resistant,non-secreting murine myeloma cell line P3X63-Ag8.653 (obtainable fromthe ATCC). The hybridoma cell lines are generally cloned by limitingdilution, and assayed for the production of antibodies which bindspecifically to the immunizing antigen and which do not bind tounrelated antigens. The selected monoclonal antibody-secretinghybridomas are then cultured either in vitro (e.g., in tissue culturebottles or hollow fiber reactors), or in vivo (e.g., as ascites inmice).

[0064] Hybridoma supernatant can be assayed for anti-MenB PS derivativereactive antibody using, for example, either solid phase ELISA or anindirect immunofluorescence assay with the immunizing MenB PS derivativeor with native MenB PS (NAc-MenB PS). The selectivity of monoclonalantibodies secreted by the hybridomas can be assessed using competitivespecific binding assays, such as inhibition ELISA, or the like. Forexample, antibody molecules, either diluted in buffer, or buffercontaining soluble MenB PS derivatives or NAc-MenB PS, are reacted in anELISA vessel in the presence of bound MenB PS derivatives. Afterwashing, bound antibody is detected by labeled anti-Ig (anti-IgM, IgGand IgA) as the secondary antibody. Antibodies that are inhibited by thesoluble MenB PS derivatives can be considered specific and, thus areselected for further study including, isotyping and additional screeningfor cross-reactivity, functional activity, and autoreactivity.

[0065] Specifically, partially purified monoclonal antibody moleculescan be individually evaluated for their ability to bind to host cellswhich express polysialic acid residues on their cell surfaces. Suchcells represent surrogate targets for the detection of antibodies thatexhibit autoimmune activity. One target comprises the humanneuroblastoma cell line, CHP-134, which expresses long chain a2-8polysialic acid (NCAM) on its cell surface, as described by Livingstonet al. (1988) J. Biol. Chem. 263:9443. Other suitable targets include,but are not limited to, newborn brain cells, tissues derived from e.g.,kidney, heart and the olfactory nerve, cultured saphenous veinendothelial cells, cytotoxic T lymphocytes and natural killer (NK)cells. See, e.g., Brandon et al. (1993) Intl. J. Immunopathology andPharmacology 6:77. Monoclonal antibody molecules obtained from thehybridomas can be added to suitable test cell populations in culture,and the potential binding of the monoclonals to the cellular targetsdetected and quantified directly using labeled monoclonals, orindirectly using an appropriately labeled secondary reagent that reactsspecifically with each monoclonal antibody (e.g., Staphylococcal ProteinA and G and anti-murine antibody molecules). Antibodies that do notcross-react with test host tissue PSA or that display minimal reactivityare not considered autoreactive for purposes of the present invention.Thus, these antibodies are appropriate for further use. In addition,some antibodies that show binding with test tissue, which binding is notaffected by pre-treatment of the test cells with neuraminidase, may alsobe appropriate for further use. Autoreactivity of such antibodies istermed “indeterminate” herein.

[0066] Functional activity can be determined by assessingcomplement-mediated bactericidal activity and/or opsonic activity. Inparticular, complement-mediated bactericidal activity of the antibodiescan be evaluated using standard assays such as those described by Goldet al. (1970) Infect. Immun. 1:479, Westerink et al. (1988) Infect.Immun. 56:1120, Mandrell et al. (1995) J. Infect. Dis. 172:1279, andGranoff et al. (1995) Clin. Diagn. Laboratory Immunol, 2:57. In theseassays, N. meningitidis is reacted with a complement source as well aswith the antibody to be tested. Secterial counts are done at varioussampling times. Those antibodies that demonstrate complement-mediatedbacterioidal activity, as demonstrated by a minimum of a 50% reductionin viable bacterial cell counts determined after sixty minutesincubation with antibody and complement, as compared to colony counts attime zero, are considered to exhibit bactericidal activity for purposesof the present invention and are suitable for further use.

[0067] Complement-mediated bacteriolysis is thought to be the majormechanism responsible for host protection against invasive Meningococcaldisease. However, evidence also supports an important protective rolefor opsonization (see, e.g., Bjerknes et al. (1995) Infect. Immun.63-169). Accordingly, the opsonic activity of the antibodies producedherein can be evalutated as a second measure, or as an alternativemeasure, to assess functional activity. Results from opsonic assays canbe used to supplement tactericidal data, and to help in the selection ofantibodies capable of conferring protection. Evaluation of optimalactivity is also particularly useful herein for the evaluation of themurine monoclonal antibodies of the invention which have an IgGlisotype. Murine IgGl (in contrast to human IgGl) is ineffective inactivation of complement. Thus, murine IgGl antibodies do not activatecomplement-mediated bacteriolysis of MenB in the above described assays.However, functional activity of IgGl anti-NPr-MenB PS monoclonalantibodies can be accessed by opsonization in the absence of complement.

[0068] A variety of opsonic assay methods are known in the art and canbe used to evaluate functional activity of the monoclonal antibodies ofthe present invention. Such standard assays include those described bySjursen et al. (1987) Acta Path. Microbiol. Immunol. Scand., Sec. C95:283, Halstensen et al. (1989) Scand. J. Infect. Dis. 21:267, Lehmannet al. (1991) APMIS 99:769, Halstensen et al. (1991) NIPH Annals 14:157,Fredlund et al. (1992) APMIS 100:449, Guttormsen et al. (1992) Infect.Immun. 60:2777, Guttormsen et al. (1993) J. Infec. Dis. 167:1314,Bjerknes et al. (1995) Infect. Immun. 63:160, Hayrinen et al. (1995) J.Infect. Dis. 171:1481, de Velasco et al. (1995) J. Infect. Dis. 172:262,and Verheul, A. F. M. (1991) “Meningococcal LPS Derived Oligosaccharide-Protein Conjugate Vaccines, Immunochemical and Immunological Aspects,”Thesis, Utrecht University, The Netherlands, pp. 112-135.

[0069] Selected monoclonal antibodies of interest can be expanded invitro, using routine tissue culture methods, or in vivo, using mammaliansubjects. For example, pristane-primed mice can be inoculated with logphase hybridoma cells in PBS for ascites production. Ascites fluid canbe stored at −70° C. prior to further purification.

[0070] It may be desirable to provide chimeric antibodies, especially ifthe antibodies are to be used in preventive or therapeuticpharmaceutical preparations, such as for providing passive protectionagainst MenB., as well as in MenB diagnostic preparations. Chimericantibodies composed of human and non-human amino acid sequences may beformed from the mouse monoclonal antibody molecules to reduce theirimmunogenicity in humans (Winter et al. (1991) Nature 349:293; Lobuglioet al. (1989) Proc. Nat. Acad. Sci. USA 86:4220; Shaw et al. (1987) JImmunol. 138:4534; and Brown et al. (1987) Cancer Res. 47:3577;Riechmann et al. (1988) Nature 332:323; Verhoeyen et al. (1988) Science239:1534; and Jones et al. (1986) Nature 321:522; EP Publication No.519,596, published 23 Dec. 1992, and U.K. Patent Publication No. GB2,276,169, published 23 Sept. 1994).

[0071] Antibody molecule fragments, e.g., F(ab₂, Fv, and sFv molecules,that are capable of exhibiting immuological binding properties of theparent monoclonal antibody molecule can be produced using knowntechniques. Inber at al. (1973) Proc. Nat. Acad. Sci. USA 69:2659;Hochman et al. (1975) Biochem 15:2706; Ehrlich et al. (1980) Biochem19:4091; Huston et al. (1986) Proc. Nat. Acad. Sci. USA 85:(16):5879;and U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and4,946,778, to Ladner et al.

[0072] In the alternative, a phage-display system can be used to expandthe monoclonal antibody molecule populations in vitro. Salkl, et al.(1986) Nature 324:163; Scharf et al. (1985) Science 133:1076; U.S. Pat.Nos. 4,683,195 and 4,683,202; Yang et al. (1995) J Mol Biol 254:392;Barbus, III et al. (1995) Methods: Comp. Meth Enzymol 8:94; Barbas, IIIet al. (1991) Proc Natl Acad Sci USA 88:7978.

[0073] Once generated, the phage-display library can be used to improvethe immunological binding affinity of the Fab molecules using knowntechniques. See, e.g., Fagina et al. (1994J. Mol. Biol. 239:68.

[0074] The ongoing sequences for the heavy and light chain portions ofthe Fab molecules selected from the image display library can beisolated or synthesized, and cloned into any suitable vector or repliconfor expression. Any suitable expression system can be used, includingfor example, bacterial, yeast, insect, amphibial mammalist systems.Expression systems in bactelia includle those described in Chang et al.(1978) Nature 275:615, Goediel et al. (1979) Nature 281:544, Goediel etal. (1980) Nucleic Acids Res. 8:4057, European Application No. EP36,776, U.S. Pat. No. 4,551,433, deBoer et al. (1983) Proc. Natl. Acad.Sci. USA 80:21-25, and Siebenlist et al. (1980) Cell 20:269.

[0075] Expression systems in yeast include those described in Hinnen etal. (1978) Proc. Natl. Acad. Sci. USA 75:1929, Ito et al. (1983) J.Bacteriol. 153:163, Kurtz et al. (1986) Mol. Cell. Biol. 6:142, Kunze etal. (1985) J. Basic Microbiol. 25:141, Gleeson et al. (1986) J. Gen.Microbiol. 132:3459, Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302,Das et al. (1984) J. Bacteriol. 158:1165, De Louvencourt et al. (1983)J. Bacteriol. 154:737, Van den Berg et al. (1990) Bio/Technology 8:135,Kunze et al. (1985) J. Basic Microbiol. 25:141, Cregg et al. (1985) Mol.Cell. Biol. 5:3376, U.S. Pat. Nos. 4,837,148 and 4,929,555, Beach et al.(1981) Nature 300:706, Davidow et al. (1985) Curr. Genet. 10:380,Gaillardin et al. (1985) Curr. Genet. 10:49, Ballance et al. (1983)Biochem. Biophys. Res. Commun. 112:284-289, Tilburn et al. (1983) Gene26:205-221, Yelton et al. (1984) Proc. Natl. Acad. Sci. USA81:1470-1474, Kelly et al. (1985) EMBO J. 4:475479; European ApplicationNo. EP 244,234, and International Publication No. WO 91/00357.

[0076] Expression of heterologous genes in insects can be accomplishedas described in U.S. Pat. No. 4,745,051, European Application Nos. EP127,839 and EP 155,476, Vlak et al. (1988) J. Gen. Virol. 69:765-776,Miller et al. (1988) Ann. Rev. Microbiol. 42:177, Carbonell et al.(1988) Gene 73:409, Maeda et al. (1985) Nature 315:592-594,Lebacq-Verheyden et al. (1988) Mol. Cell. Biol. 8:3129, Smith et al.(1985) Proc. Natl. Acad. Sci. USA 82:8404, Miyajima et al. (1987) Gene58:273, and Martin et al. (1988) DNA 7:99. Numerous baculoviral strainsand variants and corresponding permissive insect host cells from hostsare described in Luckow et al. (1988) Bio/Technology 6:47-55, Miller etal. (1986) GENERIC ENGINEERING, Setlow, J. K. et al. eds., Vol. 8,Plenum Publishing, pp. 277-279, and Maeda et al. (1985) Nature315:592-594.

[0077] Mammalian expression can be accomplished as described in Dijkemaet al. (1985) EMBO J. 4:761, Gorman et al. (1982) Proc. Natl. Acad. Sci.USA 79:6777, Boshart et al. (1985) Cell 41:521, and U.S. Pat. No.4,399,216. Other features of mammalian expression can be facilitated asdescribed in Ham et al. (1979) Meth. Enz. 58:44, Barnes et al. (1980)Anal. Biochem. 102:255, U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762,4,560,655 and Reissued U.S. Pat. No. RE 30,985, and in InternationalPublication Nos. WO 90/103430, WO 87/00195.

[0078] Any of the above-described antibody molecules can be used hereinto provide anti-MenB therapeutic or preventive pharmaceutical agents.Additionally, “humanized” antibody molecules, comprising antigen-bindingsites derived from the instant murine monoclonal antibodies, can beproduced using the techniques described above.

[0079] The anti-MenB antibodies of the present invention, describedabove, are conveniently used as receptors to screen diverse molecularlibraries in order to identify molecular mimetics of unique epitopesfrom MenB. Methods for identifying mimetics in molecular librariesgenerally involve the use of one or more of the following procedures:(1) affinity purification with an immobilized target receptor; (2)binding of a soluble receptor to tethered ligands; and (3) testingsoluble compounds directly in antigen competition assays or forbiological activity. Molecules screened for molecular mimics include butare not limited to small organic compounds, combinatorial libraries oforganic compounds, nucleic acids, nucleic acid derivatives, saccharidesor oligosaccharides, peptoids, soluble peptides, peptides tethered on asolid phase, peptides displayed on bacterial phage surface proteins,bacterial surface proteins or antibodies, and/or peptides containingnon-peptide organic moieties.

[0080] For example, libraries of diverse molecular species can be madeusing combinatorial organic synthesis. See, e.g., Gordon et al. (1994)J. Med. Chem. 37:1335. Examples include but are not limited tooligocarbamates (Cho et al. (1993) Science 261:1303); peptoids such asN-substituted glycine polymers (Simon et al. (1992) Proc. Natl. Acad.Sci. USA 89:9367); and vinylogous polypeptides (Hagihara et al. (1992)J. Am. Chem. Soc. 114:6568).

[0081] A variety of approaches, known in the art, can be used to trackthe building blocks as they are added during synthesis so that thehistory of individual library members can be determined. Theseapproaches include addressable location on a photolithographic chip(oligocarbamates), a deconvolution strategy in which “hits” areidentified through recursive additions of monomers to partiallysynthesized libraries (peptoids, peptides), and coding combinatoriallibraries by the separate synthesis of nucleotides (Nielsen et al.(1993) J. Am. Chem. Soc. 115: 9812) or other organic moieties (Ohlmeyeret al. (1993) Proc. Natl. Acad. Sci. USA 90:10922) (“tags”). The codedtags associated with each library member can then be decoded after amimetic has been selected. For example, nucleic acid tags can be decodedby DNA sequencing.

[0082] Peptoid combinatorial libraries are particularly useful foridentifying molecular mimetics of unique MenB epitopes. Peptoids areoligomers of N-substituted glycine (Simon et al. (1992) Proc. Natl.Acad. Sci. USA 89:9367) and can be used to generate chemically diverselibraries of novel molecules. The monomers may incorporate t-butyl-basedside-chain and 9-fluorenylmethoxy-carbonyl α-amine protection. Theassembly of monomers into peptoid oligomers can be performed, forexample, on a solid phase using the “submonomer method” of Zuckermann etal. (1991) J. Am. Chem. Soc. 114:10646. In this method, syntheses areconducted with Rink amide polystyrene resin (Rink et al. (1987)Tetrahedron Lett. 28:3787). Resin-bound amines are bromoacetylated by insitu activation of bromoacetic acid with diisopropylcarbodiimide.Subsequently, the resin-bound bromoacetamides are displaced by additionof an amine. The amines may incorporate t-butyl-based protection ofadditional reactive groups. This two-step cycle is repeated until thedesired number of monomers is added. The oligopeptide is then releasedfrom the resin by treatment with 95% trifluroacetic acid/5% water. Thesyntheses are performed, preferably, using a robotic synthesizer. See,e.g., Zuckermann et al. (1992) Pept. Protein Res. 40:498. In thealternative, oligomerization of the peptoid monomers may be performed byin situ activation by eitherbenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorphosphate orbromotris(pyrrolidino)phosphonium hexafluorophosphate. In thisalternative method, the other steps are identical to conventionalpeptide synthesis using α-(9-fluorenylmethoxycarbonyl) amino acids (see,e.g., Simon et al. (1992), supra).

[0083] Once the peptoid libraries are generated, they can be screenedby, e.g., adding the monoclonal antibodies of the present invention,along with various pools of the combinatorial peptoids, to wells ofmicrotiter plates coated with MenIB PS derivatives or MenB bacteria,either alone or as glycoconjugates. After a period of incubation and awash to remove unbound antibody, the presence of bound antibody isdetermined by standard ELISA assays. See, e.g., Harlow & Lane,Antibodies: A Laboratory Manual (1988), Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 553. Wells that do not contain bound antibodyindicate the presence of peptoid mimetics that bind to the antibody. Theparticular identities of the peptoid mimetics in the pools aredetermined by recursively adding back monomer units to partiallysynthesized members of the libraries. Zuckermann et al. (1994) J. Med.Chem. 37:2678.

[0084] Peptide libraries can also be used to screen for molecularmimetics of unique epitopes of MenB using the anti-MenB antibodies ofthe present invention. Such libraries are based on peptides such as, butnot limited to, synthetic peptides that are soluble (Houghten (1985)Proc. Natl. Acad. Sci. USA 82:5131) or tethered to a solid support(Geysen et al. (1987) Immunol. Methods 102:259; U.S. Pat. No. 4,708,871)and peptides expressed biologically as fusion proteins (Scott et al.(1990) Science 249:386). For a review of peptide combinatoriallibraries, see, e.g., Gallop et al. (1994) J. Med. Chem. 37:1233.

[0085] For example, random soluble peptides, having known sequences, canbe synthesized on solid supports and members of the library separatedfrom each other during the repetitive coupling/deprotection cycles inindividual labeled polypropylene bags (Houghten (1985) Proc. Natl. Acad.Sci. USA 82:5131). Following synthesis, the peptides are cleaved fromthe solid support and identified by the label on the polypropylene bag.The synthetic peptide library generated using this method can bescreened for binding to an antibody having the desired properties byadsorbing individual peptides to microtiter plate wells and determiningantibody binding using standard ELISA assays.

[0086] Large, libraries of potential peptide mimetics can also beconstructed by concurrent synthesis of overlapping peptides as describedin U.S. Pat. No. 4,708,871. to Geyser. The synthetic peprides can betested for interaction with the autibodies by ELISA while still attachedto the support used for synthesis. The solid support is generally apolyethylene or polypropylene rod onto which is graft polymerized avinyl monomer containing at least one functional group to producepolymeric chains on the carrier. The functional groups which aresequentially reacted with amino acid residues in the appropriate orderto build the desired synthetic peptide using conventional methods ofsolid phase peptide chemistry. For example, peptide sequences can bemade by parallel synthesis on polyacrylic acid-grafted polyethylene pinsarrayed in microtiter plates, as described in Geyser et al. (1987) J.Immunol. Methods 102:259. Such libraries can be screened by, e.g.,adding antibody to wells containing the peptide-pins. After washingunbound antibody from the cells, the presence of bound antibody can bedetected using an ELISA assay.

[0087] Peptide mimetics that interact with the antibodies of the presentinvention can also be identified using biological expression systems.See, Christal et al. (1992) J. Mol. Biol. 227:711; Science 249:404;Cwirla et al. (1990) Proc. Acad. Sci. USA 87:6378; Gallop et al. (1994)J. Med. Chem 37:1233. Using such systems, large libraries of peptidesequences can be screened for molecules that bind the antibodies of thepresent invention. This approach also allows for simple molecularcharacterization of identified mimetics since DNA encoding the peptidescan be readily sequenced. Additionally, rare mimetics can be amplifiedthrough several rounds of selection/amplification.

[0088] For example, phage-display libraries can be produced by insertingsynthetic DNA pieces, encoding random peptide sequences, near the 5′-endof the gene encoding the pIII or pVIII protein of the filamentousbacterial phage m13, fd, or f1 (Parmley et al. (1988) Gene 73:305; Smithet al. (1993) Meth. Enzymol. 217:228). The phage, phagemid, or plasmidDNA containing the gene and randomized extension is then used totransform a suitable host such as E. coli or E. coli coinfected with ahelper phage. The phage isolated from the culture carry pIII (1-5copies) or pVIII (˜4000 copies) surface proteins having the randomizedpeptide sequences extending from the amino terminus. Phage can bepurified by, e.g., affinity purification by biotinylating the receptorantibodies of the present invention, incubating the phage with thebiotinylated receptor and reacting the phage on streptavidin-coatedplates. Bound phage are eluted and amplified by infecting a suitablehost on agar medium and subjected to further rounds of affinitypurification. Phage from later rounds of affinity purification can becloned and propagated, their DNAs sequenced to determine the amino acidsequences of their expressed peptide and their binding to MenBantibodies assessed by ELISA or by a variety of other screeningprocedures, well known in the art.

[0089] Combinatorial libraries of human Fab antibodies can also bedisplayed on phage surface proteins to select useful molecular mimeticsfor use herein. Preparation of such libraries has been describedhereinabove. See, e.g., Burton et al. (1994) Adv. Immunol. 57:191 for areview of such techniques.

[0090] Molecular mimetics of MenB unique epitopes can also be identifiedusing the anti-MenB antibodies of the present invention in those methodsdescribed by Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865. TheCull technique utilizes the DNA binding protein, LacI, to form a linkbetween peptide and its encoding DNA sequence. In this method, DNAencoding randomized peptides is appended to the 3′-end of the LacI genepresent on a plasmid. The plasmid also contains the DNA binding site forLacI, laco. When Lacd is expressed from the plasmid in a suitable host(e.g. E. coli), it binds tightly to laco. Thus, when the cells arelysed, each copy of LacI that displays a randomized peptide at itscarboxyl terminus is associated with the DNA encoding it. Methods forscreening, amplifying, and sequencing these “peptides-on-plasmids”libraries are the same as those used in phage display, as describedabove.

[0091] Molecular mimetics can also be identified using the anti-MenBantibodies in in vitro, cell-free systems such as the system describedby Mattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022. In thisapproach, nascent peptides are displayed in polysome complexes andconstruction of libraries, expression of the peptides, and screening iscarried out in a cell-free system. Peptides displayed on polysomes canbe screened using, for example, an affinity purification/amplificationscreening procedure where the MenB-specific antibody/receptor isimmobilized, e.g., on a plastic plate.

[0092] Molecules used in the libraries above can be manipulated in orderto form more stable conformations and thus enhance identification ofuseful molecular mimetics. For example, cysteine residues can beincorporated in the randomized sequences to form disulfide loops (O'Nealet al. (1992) Proteins 14:509) and protein scaffolds can be used todisplay randomized peptides in internal loop segments (Freimuth et al.(1990) J. Biol. Chem. 265:896; Sollazzo et al. (1990) Prot. Engin.4:215).

[0093] Anti-idiotypic antibodies can also be produced using theanti-MenB antibodies of the present invention for use as molecularmimetics of unique epitopes of MenB. For a review of anti-idiotypeantibodies, see, e.g., Kieber-Emmons et al. (1986) Int. Rev. Immunol.1:1. In this regard, the pocket or cleft formed by the heavy and lightchains of an antibody is often intimately involved in antigen binding.This region, called the paratope, is an “internal image” of the antigensurface bound by the antibody. An antibody directed against the paratopeis one of several potential anti-idiotypic antibodies and can be amimetic of the antigen. Randomized peptide loops of the heavy and lightchains occur naturally as part of the generation of antibody diversity.

[0094] Anti-MenB monoclonal antibodies of the present invention can beused to elicit anti-idiotype antibody production and to selectanti-idiotypes bearing the “image” of the antigen, using the techniquesdescribed in e.g., Westerink et al. (1988) Infect. Immun. 56:1120.

[0095] In one embodiment, a combinatorial library of phage-displayantibodies, as described above, are screened using the anti-MenBmonoclonal antibodies of the present invention to identify mimeticantibodies, i.e. phage-display Fab anti-idiotypic antibodies.

[0096] Anti-idiotype antibodies produced can be easily tested for theirability to elicit anti-MenB antibody production in standard laboratoryanimal models. The variable genes of the anti-idiotype antibodies can besequenced to identify peptide vaccine candidates.

[0097] Additionally, combinatorial libraries of oligonucleotides (DNA,RNA, and modified nucleotides) can be screened to find molecularmimetics that bind to the non-autoreactive, anti-MenB antibodies of thepresent invention. Techniques for the production and use of suchlibraries are reviewed in e.g., Gold et al. (1995) Annu. Rev. Biochem.64:763. A system, known as SELEX for Systematic Evolution of Ligands byEponential enrichment, can be used for rapidly screening vast numbers ofoligonucleotides for specific sequences that have desired bindingaffinities and specificities toward the anti-MenB antibodies. (Tuerk etal. (1990) Science 249:505). For example, immobilized non-autoreactiveMenB monoclonal antibodies can be used to affinity purify specificbinding oligonucleotides from a combinatorial library. The boundoligonucleotides are released from the immobilized antibodies by addinga competitive ligand or lowering the pH. The released oligonucleotidesare either amplified directly using the polymerase chain reaction orconverted to double stranded DNA using reverse transcriptase (Tuerk etal., 1990, supra). This is followed by additional rounds of selectionand amplification until the desired mimetic is obtained. The sequencesof the oligonucleotide mimetics are determined by DNA sequencing.

[0098] Once putative molecular mimetics are identified, they are testedfor their ability to elicit functionally active (e.g., bactericidaland/or opsonic) antibodies which lack autoreactivity or have minimalautoreactivity, as described above. Molecular mimetics that have theseproperties are appropriate for further use, for example, in vaccinecompositions.

[0099] The anti-MenB monoclonal antibodies can also be used toinvestigate the bactericidal and/or opsonic function of antibodies ofdifferent specificities, as well as to identify the molecular nature ofthe unique epitopes on the MenB bacterial surface that are notcross-reactive with host PSA. Furthermore, the anti-MenB antibodies canbe used to isolate fractions of MenB bacteria or MenB PS derivatives.Once isolated, the critical epitopes reactive with the anti-MenBantibodies can be characterized and employed directly in oligosaccharideprotein conjugate vaccines or to model synthetic saccharides or mimeticsfor use in vaccines.

[0100] Molecular mimetics identified using the functionally activeanti-MenB antibodies of the invention can be used to generate antibodyreagents for use in diagnostic assays. For example, antibodies reactivewith the molecular mimetics can be used to detect bacterial antigen inbiological samples using immunodiagnostic techniques such ascompetition, direct reaction, or sandwich type assays. Such assaysinclude Western blots; agglutination tests; enzyme-labeled and mediatedimmunoassays, such as ELISAs; biotin/avidin type assays;radioimmunoassays; immunoelectrophoresis; immunoprecipitation, and thelike.

[0101] In addition, molecular mimetics, unique (e.g., non-autoimmune)Men B epitopes identified using the molecular mimetics and anti-idmonoclonal antibodies can be used herein in vaccine compositions for theprevention of MenB disease in vaccinated subjects.

[0102] The vaccine compositions can comprise one or more of the anti-idmonoclonal antibodies, molecular mimetics or non-autoimmune epitopes ofMenB. The vaccines may also be administered in conjunction with otherantigens and immunoregulatory agents, for example, immunoglobulins,cytokines, lymphokines, and chemokines, including but not limited toIL-2, modified IL-2 (cys125→ser125), GM-CSF, IL-12, γ-interferon, IP-10,MIP1β and RANTES.

[0103] The vaccines will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,ethanol, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents of buffering sustances, and the like, may be presentis any vehicles.

[0104] Adjuvants may also be used to enhance the effectiveness of thevaccines. Adjuvants can be added directly to the vaccine compositions orcan be adminstered completely, either concurrently with or shortly aftervaccine administration. Such adjuvants include, but is not limited to:(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc. (2) oil-in-water emulsion formulations(with or without other specific immunostimulating agents such as moramylpeptides (see below or bacterial cell wall components), such as for90/14837), containing 5% Squalene, 0.5% Tween 80, 0.5% Span 85(optionally containing various amounts of MTP-PE (see below), althoughnot required) formulated into submicron particles using a microfluidizersuch as Model 110Y microfluidizer (Microfludics, Newton, Mass.), (b)SAF, containing 10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymerL121, and thr-MDF (see below) either microfluidized into a submicronemulsion or vortexed to generate a larger particle size emulsion, and(c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.)containing 2% Squalene, 0.2% Tween 80, 5% one or more bacterial cellwell skeleton (CWS), preferably MPL+CWS (Detox™); (3) seponin adjuvania,such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may be usedor particle generated therefrom such as ISCOMs (Immunostimulatingcomplexes). In Freund's Complete Adjuvant (FCA) and Freund's IncompleteAdjuvant (FICA); (5, cytokines, such as interleukins (IL-1 IL-2, etc.),macrophage colony stimulating factor (M-CSF). tumor necrosis factor(TNF), etc.; and (6) other substances that act as immunostimulatingagents to enhance the effectiveness of the composition.

[0105] Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

[0106] In order to enhance the effectiveness of vaccine compositionsformed from a molecular mimetic, it may be necessary to conjugate themimetic to a carrier molecule. Such carrier molecules will notthemselves induce the production of harmful antibodies. Suitablecarriers are typically large, slowly metabolized macromolecules such asproteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, lipid aggregates (such asoil droplets or liposomes), inactive virus particles, CRM₁₉₇ (a nontoxicmutant diphtheria toxin), and the like. Such carriers are well known tothose of ordinary skill in the art. The mimetic conjugates are selectedfor their ability to express epitopes that closely resemble those foundon the surface of MenB bacterial cells. Suitable conjugates thus elicitthe formation of antibodies that have functional activity againstbacteria, and do not cross-react, or are minimally cross-reactive withpolysialic acid in host tissue as determined using the binding assaysdescribed herein.

[0107] Typically, the vaccine compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation also may be emulsified or encapsulatedin liposomes, or adsorbed to particles for enhanced adjuvant effect, asdiscussed above.

[0108] The vaccines will comprise an effective amount of the anti-idmonoclonal antibody; molecular mimetic, peptide molecular mimetic orcomplexes of proteins; or nucleotide sequences encoding the same, andany other of the above-mentioned components, as needed. By “an effectiveamount” is meant an amount of a molecule which will induce animmunological response in the individual to which it is administered andposes a minimal risk of stimulating an autoimmune response in theindividual. Such a response will generally result in the development inthe subject of a secretory, cellular and/or antibody-mediated immuneresponse to the vaccine. Usually, such a response includes but is notlimited to one or more of the following effects; the production ofantibodies from any of the immunological classes, such asimmunoglobulins A, D, E, G or M; the proliferation of B and Tlymphocytes; the provision of activation, growth and differentiationsignals to immunological cells; expansion of helper T cell, suppressor Tcell, and/or cytotoxic T cell and/or T T cell populations.

[0109] Once formulated, the vaccines are conventionally administeredparenterally, e.g., by injection, either subcutaneously orintramuscularly. Additional formulations suitable for other modes ofadministration include oral and pulmonary formulations, suppositories,and transdermal applications. Dosage treatment may be a single doseschedule or a multiple dose schedule.

[0110] Polynucleotides encoding DNA or RNA mimetics of the MenB PS canalso be used in vaccines for nucleic acid immunization. In thealternative, polynucleotides encoding peptide mimetics can be used innucleic acid immunization. Such methods generally comprise theintroduction of a polynucleotide encoding one or more of the desiredmolecules into a host cell, for the in vivo expression of the nucleicacid molecules or proteins. The polynucleotide can be introduceddirectly into the recipient subject, such as by injection, inhalation orthe like, or can be introduced ex vivo, into cells which have beenremoved from the host. In the latter case, the transformed cells arereintroduced into the subject where an immune response can be mountedagainst the molecule encoded by the polynucleotide. Methods of nucleicacid immunization are known in the art and disclosed in e.g.,International Publication No. WO 93/14778 (published 5 Aug. 1993);International Publication No. WO 90/11092 (published 4 Oct. 1990); Wanget al. Proc. Natl. Acad. Sci. USA (1993) 90:4156; Tang et al. Nature(1992) 356:152; and Ulmer et al. Science (1993) 259:1745. Generally, thepolynucleotide is administered as a vector which has been encapsulatedin a liposome and formulated into a vaccine composition as describedabove.

[0111] The anti-MenB monoclonal antibodies of the present invention, andfunctional equivalents thereof, can be used in pharmaceuticalcompositions to treat and/or prevent MenB and E. coli Kl disease inmammals. Such disease includes bacterial meningitis and sepsis, ininfants, children and adults. In this regard, the administration of ahighly-active, anti-MenB monoclonal antibody preparation to anindividual who is at risk of infection, or who has been recently exposedto the agent will provide immediate passive immunity to the individual.Such passive immunizations would be expected to be successful in bothnormal and immunocompromised subjects. Further, administration of suchmonoclonal antibody compositions can be used to provide antibody titerto MenIB in a mammalian subject, either alone, or in combination withknown anti-MenB therapeutics.

[0112] The pharmaceutical compositions of the present inventiongenerally comprise mixtures of one or more of the above describedanti-MenB monoclonal antibodies, including Fab molecules Iv fragments,sFv molecules and combinations thereof. The compositions can be used toprevent MenB disease or to treat individuals following MenB infection.

[0113] Therapeutic uses of the pharmaceutical compositions involve bothreduction and/or elimination of the MenB infection agent from infectedindividuals, as well as the reduction and/or elimination of thecirculating MenB agent and the possible spread of the disease.

[0114] As described above in regard to the vaccine compositions of thepresent invention, the pharmaceutical compositions can be adminstered inconjunction with ancillary immunoregularary agents such as IL-2,modified IL-2 (cyc125-ser125), GM-CSF, IL-12, interferon, IP-10, MIP1βand RANIES.

[0115] The preparation of pharmaceutical composition containing or moreantiodies, antibody fragments, sFv molecules or combinations thereof, asthe active ingredient (either subcutaneously, intravenously orintramuscularly). Additional forminations suitable for other modes ofadminstration include oral and pulmonary formulstions, supposteries, andtransdermal applications.

[0116] The pharmaceutical compositions are administered to the subjecton be treated in a manner compatible with the dosage formulation and inan amount that will be prophylactically and/or therapueticallyeffective. The amount of the composition to be delivered, generally inthe range of from about 50 to about 10,000 micrograms of active agentper dose, depends on the subject to be treated, the capacity of thesubject's immune system to mount its own immune-responses, and thedegree of protection desired. The exact amount necessary will varydepending on the age and general condition of the individual to betreated, the severity of the condition being treated and the mode ofadministration, among other factors. An appropriate effective amount canbe readily determined by one of skill in the art. Thus, “an effectiveamount” of the pharmaceutical composition will be sufficient to bringabout treatment or prevention of MenB disease symptoms, and will fall ina relatively broad range that can be determined through routine trials.

[0117] In addition, the pharmaceutical compositions can be given in asingle dose schedule, or preferably in a multiple dose schedule. Amultiple dose schedule is one in which a primary course ofadministration may be with 1-10 separate doses, followed by other dosesgiven at subsequent time intervals needed to maintain or reinforce theaction of the compositions. Thus, the dosage regimen will also, at leastin part, be determined based on the particular needs of the subject tobe treated and will be dependent upon the judgement of the reasonablyskilled practitioner.

III. Experimental

[0118] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0119] Efforts have been made to ensure accuracy with respect to numbersused (e.g., amounts, temperatures, etc.), but some experimental errorand deviation should, of course, be allowed for.

EXAMPLE 1 Preparation of “Sized” Glycoconjugates

[0120] An exemplary NPr-MenB oligosaccharide-tetanus toxoid conjugatevaccine, hereinafter referred to as CONJ-2, was prepared as follows. TheN-acetyl groups of MenB B polysaccharide were removed by heating thepolysaccharide to 110° C. in 2M NaOH for 6 hours in the presence ofNaBH₄. The de-acetylated polysaccharide was exhaustively dialyzed insaturated sodium bicarbonate buffer then stirred with an excess ofpropionic anhydride for 12 hours at ambient temperature. The solutionwas exhaustively dialyzed in water and the N-propionylated meningococcalB (NPr-MenB PS) polysaccharide was recovered by lyophilization.

[0121] For preparation of the conjugate vaccine, the NPr-MenBpolysaccharide was partially hydrolyzed in 10 mM sodium acetate at pH5.5 at 50° C. for 2 hours. The resulting mixture of oligosaccharides wasfractionated on Q-Sepharose. Oligosaccharides having an average degreeof polymerization (Dp) of 2-6 were first eluted with 100 mM NaCl anddiscarded. Intermediate-sized oligosaccharides were eluted with 500 mMNaCl. It was subsequently determined by analytical ion exchangechromatography using a MonoQ column that the intermediate-sizedoligosaccharides ranged in size from Dp 13 to 20 (Mean=Dp 13).

[0122] A terminal aldehyde group was generated at the non-reducing endof the intermediate-sized oligosaccharides by reacting them with 100 mMsodium periodate for 15-30 minutes at ambient temperature in the dark.Excess ethylene glycol was used to quench the oxidative reaction and theproduct was desalted on a Sephadex G-25 column. Theoligosaccharide-protein conjugate was prepared by stirring a mixture ofterminal aldehyde containing NPr MenB oligosaccharide with tetanustoxoid (molar ratio of 200:1, respectively) in 0.75 M potassiumphosphate buffer, pH 9.0 with 40 mg/ml of sodium cyanoborohydride forone day at 400C and two days at ambient temperature. The resultantNPr-MenB oligosaccharide-tetanus toxoid conjugate (CONJ-2) was finallypurified by gel permeation chromatography on Sephadex G-100 using 50 mMsodium phosphate, pH 7.0, 150 mM sodium chloride as the eluting buffer.Sialic acid and protein compositions of the conjugate vaccine weremeasured by the Svennerholm resorcinol reaction (Svennerholm, L. (1957)Biochim. Biophys. Acta. 24:604) and Lowry assays, respectively. On aweight basis, the final saccharide-to-protein ratio of the CONJ-2conjugates ranged from 0.10 to 0.25.

EXAMPLE 2 Characterization of the Glycoconjugates

[0123] The CONJ-2 glycoconjugate was characterized as follows. In orderto demonstrate covalence (e.g., establishing a covalent linkage betweenthe NPr-MenB OS and the protein carrier), a number of physico-chemicaltechniques can be used, including: SDS-PAGE; Western Blot; SephadexG-100 gel filtration; or the like. For the purposes of the presentstudy, SDS-PAGE was used to establish covalent attachment of theNPR-MenB OS/TT CONJ-2 glycoconjugates by revealing a shift to highermolecular weight for the conjugate band as compared to the carrierprotein band, per se. Western blot analysis of the CONJ-2glycoconjugates demonstrated covalence by the coincidence of positiveimmunoreactive signals for TT and NPr-MenB PS with specific anti-TT andanti-NPr-MenB PS antisera.

[0124] Based on steric factors, the use of oligosaccharides instead oflarge molecular weight polysaccharides in the preparation of the CONJ-2glycoconjugates allows for higher coupling efficiency of saccharideantigens onto the protein carrier molecule. The finalsaccharide-to-protein ratio of these NPr-MenB oligosaccharide-basedconjugates range from about 0.10 to 0.25 which corresponds to about 3 to5 NPr-MenB oligosaccharide chains covalently bound per protein carrier.On a per weight basis, the CONJ-2 glycoconjugates appear to have ahigher saccharide loading than a previously reported NPr-MenBpolysaccharide-based conjugate (U.S. Pat. No. 4,727,136) wherein CONJ-2contains, on the average, about 7.5 to 18.8 times more saccharide (using10,000 Daltons as the molecular weight of NPr-MenB PS).

[0125] In addition, constructing the CONJ-2 glycoconjugates to havesubstantially homogenous-sized saccharide moieties of a well-definedintermediate chain length (e.g., average Dp of 10-20) is expected toresult in glycoconjugates which display more consistent immunologicalbehavior. Further, the selective end-activation (e.g., selectiveintroduction of the aldehyde group at the non-reducing terminus) of theQ-Sepharose chromatography-purified NPr-MenB oligosaccharides avoids thepossibility of cross-linked, heterogenous structures which could arisefrom the use of NPr-MenB PS molecules with “active” aldehyde groupsintroduced at both termini. In this regard, it is likely thatbi-terminally activated PS (having aldehyde groups at both ends) couldbe derived from a periodate oxidation of N-acylated MenB PS previouslyexposed to NaBH₄ during the N-deacetylation procedure.

EXAMPLE 3 Preparation of Monoclonal Antibodies

[0126] 4 to 6 week old female CD1 mice were vaccinated by ip injectionusing a composition containing an NPr-MenB OS/TT (CONJ-2) glycoconjugateantigen and (except for the last booster injection) FCA. Vaccinationswere administered at one month intervals for a total of 2 or 3 dosages(including the booster immunization). Three days prior to fusion, theprimed animals were boosted with the NPr-MenB OS/TT (CONJ-2)glycoconjugate antigen in the absence of adjuvant. The final volume ofeach dose was 0.1 ml, which contained 2.5 μg of sialic acid. After thebooster injection, the animals were splenectomized and the spleen cellswere prepared for fusion with myeloma cells.

[0127] Approximately one week before fusion, non-secreting murineP3X63-Ag8.653 myeloma cells (available from the ATCC under accessionnumber ATCC-1580-CRL), were expanded in complete RPMI-1640 medium with25 mM HEPES buffer and L-Glutamine (GIBCO BRL 041-02400). The cellcultures were assessed periodically to monitor cell growth, cell numbersand to screen for contamination.

[0128] On the day of fusion, the spleen cells and the partnerP3X63-Ag8.653 myeloma cells (AgS cells) were washed, harvested and mixedat a ratio of 5:1 (spleen cells:myeloma cells). The cell fusions wereperformed at 37° C. in the presence of 50% polyethylene glycol (PEG).The resulting cell pellets were harvested and plated into 96 wellflat-bottom cell culture plates (COSTAR 3596) and incubated undersuitable conditions (e.g., at 37° C. in 5% CO₂). After one day ofincubation, selective medium containing hypoxanthine, aminopterin andthymidine (HAT) was added to each well.

[0129] Hybridomas from wells containing growing cells and exhibitingabout 10 to 25% confluence were selected for screening after about twoweeks of incubation in the HAT selective medium. Selected hybridomasupernatants were screened using a solid phase avidin-biotinylatedNPr-MenB PS based ELISA assay. Specificity of antibody binding in thesupernatants was determined using soluble NPr-MenB PS as the inhibitor.Negative controls included RPMI medium, Ag8 myeloma supernatant andirrelevant monoclonal antibody preparations. Pooled polyclonal sera frommice immunized with the NPr-MenB OS/TT (CONJ-2) glycoconjugate was usedas the positive control. After overnight incubation with thesupernatants, the reaction wells were washed and bound immunoglobulinwas detected with alkaline phosphatase-labelled polyvalent anti-murineimmunoglobulins (IgG, IgA, IgM).

[0130] Candidate hybridomas were identified based on their demonstratedbinding affinity for NPr-MenB PS in the above-described ELISA assay.Hybridomas secreting highly reactive antibody molecules were cloned bylimiting dilution. Particularly, candidate hybridoma cell lines wereplated at 0.3, 1.0 and 3.0 cell/well in Terasaki plates (NUNC) in 20 μlof cloning/expansion medium (Complete RPMI-1640 with IL6). After twoweeks, the cultures were visually inspected for growth. Frequencyanalysis was performed using the least squares method described byLefkovits et al. (1984) Immun. Today 5(9):265. The ELISA assay used toidentify reactive supernatant among the master wells was repeated toassess antibody activity on days 7 and 14. Selected clones were thenexpanded and frozen for subsequent use in tissue culture and ascitesproduction. A panel of 39 hybridomas was thus produced, and the secretedmonoclonal antibody molecules obtained therefrom (termed “SEAMmonoclonal antibodies,” particularly, monoclonal antibodies SEAM-1through SEAM-24, SEAM-26, SEAM-28 through SEAM-31, SEAM-33 throughSEAM-36, SEAM-38 through SEAM-42, and SEAM-48) were prepared for furtherevaluation.

[0131] More particularly, selected monoclonal antibodies were producedeither in tissue culture, or in ascitic fluid using Pristane-primed 7 to8 week old male Balb/c mice. Each animal subject was primed by i.p.injection with 0.5 ml Pristane one week prior to inoculation withhybridoma cells. Prior to inoculation, the hybridoma cell concentrationswere adjusted to between 2.5×10⁶ and 3×10⁶ cells/ml using sterile PBS.The primed animals were injected i.p. with 1 ml of hybridoma cells,wherein each clonal cell line was inoculated into three different mice.One to two weeks after inoculation, ascites fluid collection was startedand continued for a period of approximately one week. The collectedfluid was centrifuged at ambient temperature for 10 minutes at 2700 rpm(1500×g). Supernatants were harvested and pellets discarded. Theisolated ascites fluid was stored at 4° C. over the course ofcollection, and fluid collected on different days was pooled, aliquotedand frozen at −70° C.

EXAMPLE 4 Characterization of the Monoclonal Antibodies

[0132] The concentrations of unpurified monoclonal antibodies weredetermined using an ELISA capture assay and a radial immunodiffusionassay. Particularly, a capture ELISA procedure was used to determine theconcentration of each of the anti-NPr-Men PS monoclonal antibodies.Microtiter plates (Immulon 2, available from Dynatech Laboratories,Inc.) containing 100 μl/well of affinity purified rabbit anti-murineIgG, IgM and IgA (H and L, Zymed) diluted to 1 μg/ml in 10 mM PBS (pH7.4) were incubated overnight at 4° C. After washing three times withPBS, the wells were filled with 250 μl of Blocking Buffer (PBScontaining 1% bovine serum albumin (BSA) and 0.1% sodium azide, pH 7.4)and incubated for 30 to 60 minutes at ambient temperature to blocknonspecific binding sites. The plates were washed three times withWashing Buffer (PBS containing 0.1% Tween 20 and 0.1% sodium azide, pH7.4). Antibodies to be tested were diluted in Diluting Buffer (PBScontaining 1% BSA, 0.1% Tween 20 and 0.1% sodium azide, pH 7.4) and thenadded at 100 μl per each well. The plates were covered and incubatedovernight at 4° C. Murine IgG1, IgG2b, IgG3 and IgM immunoglobulinstandards (available from Southern Biotechnology Associates), atconcentrations ranging from 500 ng/ml to 4 ng/ml, were used to constructstandard curves for quantifying antibody concentrations.

[0133] After incubation overnight, the wells were washed five times withcold Washing Buffer and incubated for 3 hours at 4° C. with 100 μl/wellof alkaline phosphatase conjugated anti-murine IgG, IgM and IgApolyclonal antibodies (H and L, Zymed) that were diluted 1:2000 inDiluting Buffer. The plates were then washed with cold Washing Buffer,and 100 μl of freshly prepared substrate (p-Nitrophenyl phosphate,Sigma) diluted to 1 mg/ml in Substrate Buffer (1.0 M diethanolamine, 0.5mM MgCl₂, pH 9.8) was added to each well. Absorbance values at 405 nmwere measured after approximately 30 minutes. Immunoglobulinconcentrations of the monoclonal antibody preparations were calculatedfrom the standard curves.

[0134] Radial immunodiffusion assays were conducted as follows. Radialimmunodiffusion plates and reagents were obtained from The Binding SiteLimited (Birmingham, England). The assay protocol was then based on themanufacturer's specific instructions supplied with the RID kit. Briefly,calibrator antibody supplied with the kit was reconstituted with anappropriate amount of distilled water. 1:2 and 1:10 dilutions ofcalibrator antibody were prepared. Test samples can be diluted in 1% BSAif necessary. Aliquots of 10 μl (20 μl for IgA and IgG2a subclassantibodies) for calibrator antibody (neat, 1:2, and 1:10 dilutions) andtest samples were applied to separate wells on the plate and incubatedfor 120 hours at room temperature. The concentrations of the antibodieswere determined by measuring the precipitation ring diameters andcomparing these values to a reference table included with the RID kit.

[0135] The monoclonal antibodies from tissue culture or ascitic fluidwere then partially purified as follows. Tissue culture supernatant orascites containing the monoclonals (200 ml or indicated volume) wasadded slowly to an equal volume of cold 100% saturated ammonium sulfate(SIGMA, Saint Louis, Mo.) while stirring the solution gently. Themonoclonal antibody and Ammonium sulfate mixture was incubated overnightat 4° C. The following morning, the mixture was stirred gently tohomogeneity and centrifuged at 5000 rpm in a Sorvall SS34 rotor for 30minutes at 4° C. After decanting the supernatant, an equal volume of 50%ammonium sulfate solution (i.e. same volume as the 100% saturatedammonium sulfate) was used to wash and resuspend the pellet. Theresulting mixture was centrifuged at 5000 rpm in a Sorvall SS34 rotorfor 30 minutes at 4° C. The supernatant was then decanted and drained.

[0136] For ascites, the pellet was reconstituted in 0.3-0.5 volumes ofthe starting volume in PBS Buffer (50 mM sodium phosphate, 150 mM sodiumchloride, pH 7.4).

[0137] For tissue culture supernatant, the pellet was reconstituted in0.1 volumes of the starting volume of PBS Buffer. The reconstitutedmonoclonal antibody and ammonium sulfate mixture was placed in adialysis tubing (molecular weight cut off 10,000-12,000) and allowed todialyze in 4 L of PBS overnight. The PBS solution was changed 3 to 4times over the following two days. Monoclonal antibody molecules fromthe dialysis tubes were transferred into a syringe and sterile filteredthrough a 0.2 μm membrane filter, and then stored at −20° C.

[0138] The partially purified monoclonal antibody preparations were thencharacterized for (a) immunoglobulin isotype, (b)concentration-dependent binding to NPr-MenB PS, (c) the ability ofvarious NPr-MenB oligomers to inhibit binding to NPr-MenB PS, (d)cross-reactivity with native MenB PS, (e) cross-reactivity with virulentstrains of MenB, (f) complement-mediated bactericidal activity, (g)opsonic activity, and (h) autoreactivity as demonstrated by binding to aneuroblastoma cell line that expresses long chain α2-8 linked polysialicacid at the cell surface. In these experiments, the concentrations ofmonoclonal antibody were measured by the capture ELISA and RID assaydescribed above.

(a) Isotyping of the Antibodies

[0139] The isotypes of the monoclonal antibodies (heavy and lightchains) were determined by ELISA using the above-described protocol forthe anti-NPr-MenB PS ELISA with the only difference that the secondaryalkaline phosphatase-conjugated antibody was specific for IgGsubclasses, IgM, IgA and K and X light chains. A kit was also used toisotype the antibody molecules. The kit consisted of typing sticksubstrates coated with goat antibodies specific for the different typesof immunoglobulin peptide chains. The kit provides a peroxidase-labelledspecies specific for anti-murine immunoglobulin to detect the murinemonoclonal antibodies bound to the goat antibodies on the substrate.

[0140] As depicted below in Table 1, the isotypic distribution among the39 monoclonal antibodies was found to consist of one IgM andthirty-eight IgG (eight IgG1, five IgG2a, sixteen IgG2b, and nine IgG3).In addition, all antibody molecules had K light chains. TABLE 1* Bindingto Fine SEAM ELISA ELISA Inhibition ELISA Encapsulated AntigenicMonoclonal Reactivity to of N-Pr-MenB Reactivity to Neisseria Binding toOpsono- Specificity Antibody Ig N-Pr-MenB Binding by N-Pr- N-Ac-MenBmeningitidis CHP134 Bactericidal phagocytotic Group (a) Number IsotypePS (b) MenB OS (c) PS (d) group B (e) PSA (f) Activity (g) Activity (g)I 10 G1, κ + + + + + + + + + 0 ND 0 11 G2b, κ + + + + + + + + + + + + +ND 18 G2b, κ + + + + + + + + + + + + + + + + 20 G2b, κ +/− + + + + 0 0 0ND 21 G2b, κ +/− + + + + + 0 0 0 ND 26 G2b, κ + + + + + + + + + + + + +ND 28 G2b, κ + + + + + + + + + + + + + + 29 G2a, κ + + + + + + + + + + +0 ND 35 G2b, κ + + + + + + + + + + + + + + + + II 12 G2a, κ + + + +0 + + + + + + + + + 13 G3, κ + + + 0 + + + + + + + + + + + + + 14 G2b,κ + + + + 0 + + + + + + + + ND 15 G2b, κ + + + + 0 + + + + + + + + ND 16G2b, κ + + + 0 + + i + + 0 30 G3, κ + + + 0 + + + + + + + + + + + + +III 1 G3, κ + + 0 0 0 + + ND 3 G2b, κ + + + + + + + 0 + 0 + + + + + + 4G1, κ + + + + 0 i i ND ND 5 G3, κ +/− + 0 + 0 + + + 0 7 G3, κ + + 0 ii + + + 0 8 G3, κ + + + + + + 0 + 0 + + + 0 17 M, κ + + + + 0 0 0 0 ND19 G2a, κ + + + + 0 0 i 0 ND 22 G2b, κ + + + 0 0 i 0 ND 23 G2b, κ + + +0 0 0 0 ND 48 G2b, κ + + + + + + 0 + 0 + + + + IV 2 G3, κ +/− 0 0 +0 + + + 0 6 G3, κ +/− 0 0 0 i 0 ND 9 G1, κ + + 0 0 0 i ND ND 24 G2b,κ + + 0 0 + 0 0 ND ND 31 G1, κ +/− ND + + i ND ND 36 G2a, κ + + +ND + + + + + + + ND 39 G2a, κ +/− ND + + 0 + + 0 ND 40 G1, κ + + + +ND + + + + 0 ND 41 G2b, κ + + ND + + 0 + + 0 33 G1, κ + ND 0 0 0 ND ND34 G3, κ +/− ND 0 0 0 0 ND 38 G1, κ +/− ND 0 0 0 ND ND 42 G1, κ +/− ND0 + i ND ND

(b) Concentration-Dependent Binding to NPr-MenB PS

[0141] A solid phase ELISA procedure was used to assess theconcentration dependent binding of the antibody molecules to NPr-MenB PSin the presence of buffer alone or 25 μg/ml of a soluble NPr-MenB PSinhibitor. Biotinylated NPr-MenB PS-ADH was prepared using the method ofSutton et al. (1985) J. Immunol. Methods 82:215. Microtiter plates(Immulon 2, available from Dynatech Laboratories, Inc.) containing 100μl/well of avidin (4 μg/ml Extr Avidin, Sigma) in 10 mM PBS (pH 7.4)were incubated overnight at 4° C. After washing three times with PBS,100 μl of biotinylated NPr-MenB PS in PBS was added to each well andincubated at 37° C. for 2 hours. The plates were washed three times withPBS, and the wells were filled with 250 μl of Blocking Buffer andincubated for 30 to 60 minutes at ambient temperature to blocknonspecific binding sites.

[0142] After blocking, the plates were washed three times with WashingBuffer. 50 μl aliquots of various dilutions of the monoclonals wereadded to wells of replicate plates containing either 50 μl of DilutingBuffer or 50 μl of Diluting Buffer containing 50 μg of soluble NPr-MenBPS per ml (for a final inhibitor concentration of 25 μg/ml). The plateswere then covered and incubated overnight at 4° C. On the following day,the wells were washed five times with cold Washing Buffer and thenincubated for 3 hours at 4° C. with 100 μl/well of alkaline phosphataseconjugated anti-murine IgG, IgM and IgA polyclonal antibodies (Zymed)diluted 1:2000 in Diluting Buffer. The plates were then washed with coldWashing Buffer, and 100 μl of freshly prepared substrate (p-Nitrophenylphosphate, Sigma) diluted to 1 mg/ml in Substrate Buffer was added toeach well. Absorbance values at 405 nm were measured after approximately30 minutes.

[0143] FIGS. 1A-1D show the dose-response binding activity of fourrepresentative anti-NPr-MenB PS monoclonal antibodies (SEAM-3, SEAM-5,SEAM-16 and SEAM-18, respectively), to solid phase NPr-MenB PS asdetermined by ELISA. Data shown are for the antibodies diluted in buffer(), or in buffer containing 25μg/ml of soluble NPr-MenB PS (∘).Different ranges for the X axis in the data are used, wherein monoclonalantibodies SEAM-3, SEAM-16 and SEAM-18 are shown at 0.0001 to 1 μg/ml,and monoclonal antibody SEAM-5 is shown at 0.1 to 100 μg/ml. Theconcentration of antibody sufficient to yield an OD of 0.5 afterincubation with substrate varied considerably (compare binding of SEAM-5to binding of SEAM-18).

[0144] Table 1 summarizes the respective concentration ranges ofantibody required to yield an OD of 0.5 in an ELISA for each of the 39SEAM monoclonal antibodies. The most likely explanation for the largeheterogeneity in the values shown is differences in antibody avidity toNPr-MenB PS.

(c) Inhibition of Antibody Binding to NPr-MenB PS by Oligomers

[0145] A competitive solid phase ELISA procedure was used to assess theability of NPr-MenB oligomer inhibitors to inhibit binding of themonoclonal antibody molecules to solid phase NPr-MenB PS. The assay wasperformed as described above for the anti-NPr-MenB PS ELISA with theexception that the monoclonal antibodies were pre-diluted toconcentrations to yield an OD of 0.5 to 1. The monoclonal antibodieswere added to wells of replica plates, each containing one of thefollowing soluble inhibitors to yield a final inhibitor concentration of25 μg/ml: high molecular weight (HMW) NPr-MenB PS; or low molecularweight (LMW) NPr-MenB OS (having an average Dp of 3.8).

[0146] The plates were covered and incubated overnight at 4° C. On thefollowing day, the wells were washed five times with cold Washing Bufferand then incubated for 3 hours at 4° C. with 100 μl/well of alkalinephosphatase conjugated anti-murine IgG, IgM and IgA polyclonalantibodies (Zymed) diluted 1:2000 in Diluting Buffer. The plates werethen washed with cold Washing Buffer, and 100 μl of freshly preparedsubstrate (p-Nitrophenyl phosphate, Sigma) diluted to 1 mg/ml inSubstrate Buffer was added to each well. Absorbance values at 405 nmwere measured after approximately 30 minutes. Percent inhibition wascalculated as compared to binding in the absence of inhibitor.

[0147]FIG. 2 depicts the inhibition of binding of four representativeanti-NPr-MenB PS monoclonal antibodies (SEAM-2, SEAM-3, SEAM-16 andSEAM-18) to solid phase NPr-MenB PS by either 25 μg/ml of soluble highmolecular weight (HMW) NPr-MenB PS inhibitor (▪), or 25 μg/ml of lowmolecular weight (LMW) NPr-MenB oligosaccharide (average Dp of 3.8)inhibitor (□).

[0148] The HMW NPr-MenB PS inhibitor provided approximately 75% to 95%inhibition in all monoclonal antibodies tested. Differences in fineantigenic specificity in the monoclonal antibodies are evident from thedifferent respective patterns of inhibition with the LMW inhibitortested. For example, binding of SEAM-3 and SEAM-18 to NPr-MenB PS iscompletely inhibited by the soluble LMW inhibitor of NPr-MenB PS. Incontrast, SEAM-2 and SEAM-16 are not significantly inhibited by theoligomers (less than 20%). The results of LMW NPr-MenB OS inhibition forall of the monoclonal antibodies are depicted in Table 1. In addition,as described below, other differences in the fine antigenic specificityof the monoclonals are evident by the differences observed incross-reactivity to NAc-MenB PS in ELISA and differences in binding tohost polysialic acid.

(d) Cross-Reactivity with NAc-MenB PS

[0149] The monoclonal antibodies were evaluated for their ability tocross-react with the NAc-MenB polysaccharide as demonstrated by directbinding to NAc-MenB PS in a solid phase ELISA format. The method usedwas similar to that described above for the NPr-MenB PS ELISA, with theexception that NAc-MenB PS-ADH was used as the solid phase antigeninstead of biotinylated NPr-MenB PS.

[0150] 50 μl aliquots of various dilutions of the monoclonals were addedto wells of replicate plates containing either 50 μl of Diluting Bufferor 50 μl of Diluting Buffer containing 50 μg of soluble NAc-MenB PS perml (for a final inhibitor concentration of 25 μg/ml). The plates werethen covered and incubated overnight at 4° C. On the following day, thewells were washed five times with cold Washing Buffer and then incubatedfor 3 hours at 4° C. with 100 μl/well of alkaline phosphatase conjugatedanti-murine IgG, IgM and IgA polyclonal antibodies (Zymed) diluted1:2000 in Diluting Buffer. The plates were then washed with cold WashingBuffer, and 100 μl of freshly prepared substrate (p-Nitrophenylphosphate, Sigma) diluted to 1 mg/ml in Substrate Buffer was added toeach well. Absorbance values at 405 nm were measured after approximately30 minutes.

[0151]FIG. 3 depicts the binding of five representative anti-NPr-MenB PSmonoclonal antibodies (SEAM-12, SEAM-16, SEAM-18, SEAM-2, and SEAM-3) tothe solid phase NAc-MenB PS. As can be seen, three of the antibodies,SEAM-12, SEAM-16 and SEAM-18, showed significant binding when tested at0.5 and/or 5 μg/ml of antibody. Two other antibodies, SEAM-2 and SEAM-3,previously shown to be negative in a screening assay, were confirmed asnegative when tested at 5-fold higher concentrations (25 μg/ml ofantibody). The cross-reactivity of each of the 39 monoclonal antibodieswith the NAc-MenB PS was scored over a range of (+++) for highly crossreactive, to (0) for non cross-reactive. The results are depicted inTable 1. As can be seen, sixteen of the monoclonal antibodiescross-reacted with the NAc-MenB PS, and four minimally cross reacted (±)(FIG. 1). Specificity of the cross-reactivity of these twenty positive,or weakly positive monoclonal preparations was confirmed by inhibitionof binding using soluble NAc-MenB PS. The 26 non cross-reactivemonoclonal antibodies showed no significant binding to solid phaseNAc-MenB PS when tested at antibody concentrations up to 25 μg/ml.

(e) Bacterial Binding Assay

[0152] The ability of the anti-N-Pr meningococcal B polysaccharideantibodies to bind to the surface of pathogenic strains of N.meningitidis Group B was determined using flow cytometric detection ofindirect immunofluorescence assay. Two fully encapsulated meningococcalB test organisms were used, strain 8047 (the strain used to measurebactericidal activity, see below) and NmB. A third unencapsulatedstrain, M7, which is a transposon-containing mutant of NmB (Stephens etal. (1991) Infect. & Immun. 59:4097-4102) was used as a negative controlfor specificity of antibody binding to the capsular polysaccharide.Bacterial cells grown to mid-log phase in Mueller-Hinton broth and 0.25%glucose were harvested and resuspended in Blocking Buffer at a densityof ˜10⁶ cells per ml. The monoclonal antibodies (concentration of 10 or100 μg/ml) were then added and allowed to bind to the cells on ice for 2hours. Following two washes with Blocking Buffer, the cells wereincubated with FITC-conjugated F(ab′)₂ fragment goat anti-mouse IgG(H+L) (Jackson Immune Research, West Grove, Pa.), fixed with 0.25%formaldehyde in PBS buffer, and analyzed by flow cytometry.

[0153] Positive control antibodies included meningococcal-specificserotyping and subtyping monoclonal antibodies (MN2C3B, MN16C13F4, RIVM,Bilthoven, the Netherlands). The negative control consisted of a mouseIgG monoclonal antibody of irrelevant specificity.

[0154] FIGS. 4A-4G show the results from a representative experiment.Monoclonal antibodies SEAM-3 and SEAM-18 show strong capsular-specificbinding to both encapsulated test strains. (FIGS. 4C and 4D,respectively) in this indirect fluorescence flow cytometry assay. Incontrast, monoclonal antibodies SEAM-9 and SEAM-10 were negative in thisassay (FIGS. 4E and 4F). As summarized in Table 1, twenty-four of theanti-N-Pr meningococcal B polysaccharide antibodies showed evidence ofbacterial binding when tested at 100 μg/ml. Two additional antibodiesshowed evidence of minimal binding to both encapsulated andnon-encapsulated mutant strains. Bacterial binding of these antibodieswas scored as indeterminant (i). See, for example, the binding of SEAM-7depicted in FIG. 4G.

(f) Complement-Mediated Bactericidal Activity

[0155] A bactericidal assay was conducted using the methods described byMandrell et al. (1995) J. Infec. Dis. 172:1279, with the followingmodifications: the organism was grown in Mueller-Hinton broth containing0.25% glucose; and serum diluting buffer consisted of Gey's bufferinstead of barbitol buffer. In several experiments, different sources ofcomplement were used: these included two different infant rabbit serumpools (referred to as Rab C I and Rab C II) and human agammaglobulinemicserum (referred to as Hu C).

[0156] The percent survival of N. meningiditis strain 8047 whenincubated with different concentrations of antibody and 20% complementis shown for four representative monoclonal antibodies (FIGS. 5A-5D).Each antibody shown was tested with three different complement sources:infant rabbit serum pool I (▴), infant rabbit serum pool II (), andhuman agammaglobulinemia (∘). For SEAM-5 and SEAM-12, a similar doseresponse for each antibody was observed for each of the three complementsources. In contrast, SEAM-18 required higher antibody concentrations toelicit bacterial killing in the presence of human complement than wererequired with either source of rabbit complement. SEAM-3 showedeffective killing when tested with the two rabbit complement sources,and no activity with the human complement source. The ability of each ofthe monoclonal antibodies to activate complement-mediated bacteriallysis is reported in Table 1. There are examples of bactericidalantibodies that cross react with NAc-MenB PS by ELISA (e.g., SEAM-18,SEAM-30, and SEAM-35). There also are examples of bactericidalantibodies that show no cross-reactivity with NAc-MenB PS (e.g., SEAM-2,SEAM-5, SEAM-7, and SEAM-8).

(g) Onsonic Activity

[0157] Opsonic activity of the monoclonal antibodies can be measured bya variety of established methods. Sjursen et al. (1987) Acta Path.Microbiol. Immunol. Scand., Sec. C 95:283, Halstensen et al. (1989)Scand. J. Infect. Dis. 21:267, Lehmann et al. (1991) APMIS 99:769,Halstensen et al. (1991) NIPH Annals 14:157, Fredlund et al. (1992)APMIS 100:449, Guttormsen et al. (1992) Infect. Immun. 60:2777,Guttormsen et al. (1993) J. Infec. Dis. 167:1314, Bjerknes et al. (1995)Infect. Immun. 63:160, and Hayrinen et al. (1995) J. Infect. Dis.171:1481.

[0158] In one opsonization assay, N. meningitidis freshly grown on GNagar plates (Greiner Labortechniek, Greiner BV, Alphen a/d Rijn,Netherlands) at 37° C. was used to inoculate 8 ml of Mueller Hintonbroth (Difco, Detroit, Mich.) to obtain an initial OD of 0.1. Thebacteria were grown to log phase (660 nm absorbance of 0.75-0.85) withvigorous shaking. The cells were transferred to sterile plastic tubeswith caps and centrifuged for 10 minutes at 3500 rpm.

[0159] Cells were fixed by adding 4 ml of 70% ethanol and incubating forat least 1 hour 4° C. The fixed cells were again pelleted bycentrifugation for 10 minutes at 3500 rpm and resuspended in sterilephosphate buffered saline (PBS) to yield an OD of 1.0. The cellsuspension (1.35 ml) was added to an eppendorf tube and centrifuged for5 minutes at 10,000 rpm. The supernatant was discarded, and another 1.35ml was added to the same tube followed by centrifugation to yield 1×10⁹cells per tube. A 1.0 mg/ml solution of fluorescein isothiocyanate(FITC) in PBS (Sigma, St. Louis, Mo.) was prepared and sonicated for 5minutes, then centrifuged for 5 minutes at 10,000 rpm. The FITC-PBSsolution (50 μl) was added to each tube of bacteria and then incubatedfor 1 hour at 37° C. with slight agitation. PBS (950 μl) was added toeach tube and centrifuged for 2 minutes at 10,000 rpm. The pellet waswashed once with 1 ml of PBS and once with 1 ml of BSA-Hanks balancedsalt solution (BSA-HBBS). The FITC labelled meningococci werereconstituted in 1% BSA-HBBS and divided into 100 μl aliquots which werestored at −20° C. until use in the assay.

[0160] Human polymorphic nuclear cells (PMN) were isolated from theperipheral blood of healthy adults in heparin-containing tubes (BectonDickinson, Mountain View, Calif.). A volume of 10 ml of blood wasdiluted with an equal amount of phosphate buffered saline (PBS; pH 7.4)and layered on a Ficoll histopaque gradient consisting of 10 ml ofFicoll Paque™ (Pharmacia, Uppsaila, Sweden) on top of 12 ml ofhistopaque (density 1.119, Sigma Diagnostics, St. Louis, Mo.). Aftercentrifugation at 400×g for 20 minutes at room temperature, the PMN werecollected from the upper part of the histopaque and ice cold RPMI medium(Roswell Park Memorial Institute, NY) containing 1% gelatin was added.Cells were centrifuged at 250×g and the residual erythrocytes were lysedby resuspending the cells in 9 ml of ice cold distilled water. After 1minute, concentrated PBS and RPMI-gelatin was added to make the cellsuspension isotonic. The PMN were centrifuged and resuspended in RPMImedium to a density of 1×10⁷/ml. The purity and viability of the PMN wasgreater than 95%.

[0161] To a microtiter plate was added appropriate dilutions ofmonoclonal antibody to be tested (diluted in BSA-HBBS), 5 μl of 10%human complement (in BSA-HBBS), and 25 μl of FITC-labelled bacteriasuspension to yield a total volume of 50 μl. Selected antibodies weretested without complement, and with up to three different complementsources: normal pooled human serum; agammaglobulinemic serum; and infantrabbit serum, varying the complement concentration from 1 to 10%. Eachassay included a positive and negative antibody control, as well as acomplement, non-opsonization and a cells-only control. The opsonizationreaction was allowed to proceed for 30 minutes at 37° C. on a shakerbefore terminating the reaction by placing the microtiter plate on ice.

[0162] Phagocyte cell suspension (50 μl) was added to a finalconcentration of 5×10⁶ cells/ml. This gives a ratio of bacteria tophagocytes of 10:1. Phagocytosis was allowed to proceed for 30 minutesat 37° C. on a shaker, after which time it was placed on ice. ColdBSA-HBBS (100 μl) was added to each well. The plates were centrifugedfor 10 minutes at 1100 rpm. Supernatants were aspirated from the wellsand the cells were washed twice more with 150 μl of cold BSA-HBBS. ColdBSA-HBBS (150 μl) was then added, and the resulting cell suspensionswere transferred to sterile tubes. A solution of 2% paraformaldehyde(Polysciences, Inc., Warrington, Pa.) in PBS was added to fix the cells.The samples were then analyzed by indirect florescence flow cytometry.

[0163] The results of the opsonization experiments for sixteenrepresentative SEAM monoclonal antibodies are reported in Table 1. Allantibodies found to be opsonic were also bactericidal in the assaydescribed above using at least one of the complement sources. However,as can be seen in Table 1, there are examples of antibodies that werebactericidal but not opsonic (see, e.g., SEAM-2, SEAM-5, SEAM-7,SEAM-16, and SEAM-41).

(h) Evaluation of Autoreactivity

[0164] Partially purified tissue culture supernatants containing the 39SEAM monoclonal antibodies were evaluated for autoreactivity to hostpolysialic acid. In one assay, the monoclonal antibodies were assessedfor their ability to cross-react with the human neuroblastoma cell lineCHP-134 (Livingston et al. (1988) J. Biol. Chem. 263:9443) using flowcytometric detection of indirect immunofluorescence. In this assay, theCHP-134 cells, which express long chain polysialic acid (PSA) associatedwith neuronal cell adhesion molecule (NCAM) on their surface, serve ascellular markers for human PSA antigens. In control experiments, nearlyconfluent cell cultures were collected in 50 ml centrifuge tubes andcentrifuged at 1000×g. After the supernatant was decanted, 5 ml ofBlocking Buffer was added to resuspend the cells. The cells were thencounted in a hemacytometer, and divided into two equal aliquots. Onealiquot was incubated for 2 hours at ambient temperature withexoneuraminidase (10 units/10⁸ cells, SIGMA Chemical Co., Saint Louis,Mo.); the other aliquot was treated identically but without enzyme.After incubation, the cells from each aliquot were distributed amongindividual reaction tubes so that each tube contained 10⁶ cells. To washthe cells, 2 ml of Blocking Buffer was added to each reaction tube, thetubes centrifuged at 1000 rpm in a Sorvall RT-600B for 6 minutes at 20°C., and the supernatant aspirated off. The washed cells were incubatedfor 2 hours in a total volume of 200 μl on ice with either no antibody,or the indicated concentration (usually 10 or 100 μg/ml) of the testantibody (i.e., SEAM MAbs).

[0165] Control antibodies in the assay included: (1) an IgG monoclonalantibody of irrelevant specificity (VIIG10, as a negative control); (2)an IgM anti-polysialic acid monoclonal antibody (2-1B, as a positivecontrol); and (3) an anti-CD56 monoclonal antibody specific for theprotein backbone of NCAM (Immunotech, Marseille, France). BlockingBuffer (2 ml) was added to each reaction tube, and the tubes werecentrifuged at 1000 rpm in the Sorvall RT-600B for 6 minutes at 20° C.Following centrifugation, the supernatant was aspirated off and thecells incubated for 1 hour at ambient temperature with 150 μl offluorescein isothiocyanate (FITC)-conjugated F(ab′)₂ fragment goatanti-mouse IgG (H+L) (diluted to 4 μg/ml) (Jackson Immune Research, WestGrove, Pa.). After washing with Blocking Buffer, 400 μl of 0.25%formaldehyde in PBS buffer (50 mM sodium phosphate, pH 7.0, 150 mMsodium chloride) was added to the cells, and the cells were analyzed byflow cytometry using a FACSCAN™ cell sorter (Becton-Dickinson, MountainView, Calif.).

[0166] All antibodies were tested at final concentrations of 10 and 100μg/ml of antibody in replicate, using untreated cells, and cells thathad been pre-treated with neuraminidase. This treatment cleaves thesurface polysialic acid and provides a control in the assay forspecificity of antibody binding to polysialic acid. In a typicalexperiment (FIGS. 6A-6I), cells incubated without primary antibody, orwith a control monoclonal antibody having an irrelevant antigenicspecificity, show very little fluorescence (approximately 98% of thecells have <10 units of fluorescence, FIG. 6A). In contrast, virtuallyall cells treated with the anti-NAc MenB PS monoclonal antibody, 2-1B,fluoresce strongly (FIG. 6B, left). This fluorescence is decreased tocontrol levels when the antibody is incubated with cells that had beenpre-treated with neuraminidase (FIG. 6B, right). Similarly, cellstreated with anti-CD56 fluoresce strongly (FIG. 6C). With this antibody,the fluorescence is unaffected by pre-treatment of the cells withneuraminidase since the CD56 determinant is located in the proteinbackbone of NCAM and is unaffected by the removal of polysialic acidwith neuraminidase.

[0167] The SEAM-5 antibody gives no detectable binding when tested at100 μg/ml (FIG. 6D), and is considered as negative in this assay. TheSEAM-35 antibody shows strong polysialic acid-specific binding whentested at 10 or 100 μg/ml (FIGS. 6E and 6F), and is considered positive.A few anti-NPr MenB PS monoclonal antibodies show binding when tested at100 μg/ml, but appear to be negative when tested at 10 μg/ml (see, e.g.,SEAM-12 in FIGS. 6G and 6H). Such antibodies are considered minimallyautoreactive for the purposes of this application. A rare antibodyappeared to have weak reactivity with the neuroblastoma cell line thatwas unaffected by the by pre-treatment of the cells with neuraminidase(see SEAM-7, FIG. 6I). The autoreactivity of such antibodies withpolysialic acid was scored as indeterminant in the assay, and theseantibodies were also considered to have minimal autoreactivity to hostPSA for purposes of this application.

[0168] Table 1 summarizes the autoantibody activity of each antibody asdetermined in this indirect fluorescence flow cytometry assay.Cross-reactivity with polysialic acid antigens expressed in CHP-134cells was closely correlated with the cross-reactivity of the antibodieswith NAc-MenB PS in the ELISA assay. As shown in Table 1, monoclonalantibodies that did not cross react with NAc-MenB PS in the ELISA alsodid not bind to CHP-134 cells, while all of the antibodies thatcross-reacted with NAc-MenB PS in the ELISA also cross-reacted with PSA.This correlation between the two assays was not unexpected since thepolysaccharide covalent structure of NAc-MenB PS and the host PSA isreported to be the same.

EXAMPLE 5 Passive Immunization Using SEAM Monoclonal AntibodyCompositions

[0169] In order to assess the ability of the above-characterized SEAMmonoclonal antibodies to provide passive protection against bacterialchallenge, the following immunization study was carried out.

[0170] Animals: Outbred infant SPF (specific pathogen-free) albinoWistar rats were obtained from the Helsinki University Animal Center(Helsinki, Finland).

[0171] Bacterial Strains: Neisseria meningitidis group B strain IH 5341,a human patient isolate with MenB:15:p1.7, 16 phenotype, plus 1 to 2additional other group B bacterial strains (e.g. M355; B:15:P1.15) wereused. All bacteria strains were rat passaged five times and stored inskim milk at −70° C. For each experiment, a fresh inoculum was takenfrom the stock and cultivated on gonococcal (GC) medium base (GC-agar IIBase, Becton Dickinson, Mountain View, Calif.) supplemented with.IsoVitaleX, L-tryptophan and hemoglobin. After incubation overnight at37° C. in 5% CO₂, several colonies were inoculated into a culture flaskcontaining 20 ml of brain-heart infusion broth and incubated at 37° C.in a rotatory shaker at 150 rpm until the optical density (Klett 90)corresponded to 10⁸ cfu/ml. The cultures were then diluted in phosphatebuffered saline (PBS) corresponding to 10⁶ cfu/ml for use. The actualnumber of viable bacteria in a challenge dose was determined by countingthe cfu after serial dilution of the suspension in PBS and plating onproteose peptone agar.

[0172] Immunizations: In each experiment 3-4 litters of 4-6 day oldinfant rats were randomly selected and divided into experimental groupsof 6 animals each and injected intraperitoneally with either a SEAMmonoclonal antibody composition (in 0.9% saline), saline solution(0.9%), or control antibodies. In each group, three animals wereinoculated with the SEAM antibodies (at doses of 0.4μg, 2 μg, and 10 μg,respectively), two animals were used as negative controls (one receivedinjection with saline alone while the other received injection with amonoclonal antibody of irrelevant specificity), and a positive animalreceived an injection of an anti-Men B polysaccharide antibody.

[0173] Bacterial Challenge: One to two hours after the initialinjection, the infant rats received a bacterial challenge injectionintraperitoneally of 10⁵ Neisseria meningitidis group B bacteria of thestrain IH 534 (rat passaged five times) in a final volume of 100 μl. Sixhours after bacterial inoculation, bacteremia and meningitis developmentwas assessed by culturing blood and cerebrospinal samples taken from theinfant rats.

[0174] The results of the study (protection from N. meningitidisbacteremia) for six representative SEAM monoclonal antibodies (SEAM-5,SEAM-7, SEAM-8, SEAM-10, SEAM-12, and SEAM-18) are depicted below inTable 4. As can be seen, the SEAM-12 and SEAM-18 antibodies are stronglyprotective, the SEAM-7 and SEAM-8 antibodies partially protective, withthe SEAM-5 and SEAM-10 antibodies providing no protection up to a doseof 10 μg/pup. TABLE 2 Blood Titer in cfu/ml × 10⁵ (% of Cerebral Bloodnegative Spinal Fluid SEAM Mab (positives/all) control) (positives/all)Dose: 10 μg/pup  5 5/6  0.63 (31%) 3/6  7 0/6 <0.01 (<1%) 0/6  8 1/6<0.01 (<1%) 0/6 10 6/6 10.67 4/6 (>100%) 12 0/6 <0.01 (<1%) 0/6 18 0/6<0.01% (<1%)    0/6 Dose: 2 μg/pup  5 6/6  0.37 (18%) 2/6  7 4/6  0.04(<1%) 0/6  8 6/6   2.52 (>100%) 4/6 10 6/6 10.35 5/6 (>100%) 12 1/6 0.01 (<1%) 1/6 18 1/6 <0.01% (<1%)    1/6 Dose: 0.4 μg/pup  5 5/5  5.65 (>100%) 4/5  7 6/6   9.28 (>100%) 5/6  8 6/6  1.50 (63%) 4/6 106/6 10.67 4/6 (>100%) 12 6/6  9.51 (76%) 5/6 18 5/5  3.51% 3/5 (>100%)

EXAMPLE 6 Identification of Peptide Mimetics of MenB Antigen Using SEAMMonoclonal Antibodies

[0175] The following procedures were carried out in order to identifypeptide mimetics that interact with the SEAM monoclonal antibodies ofthe present invention. Phage display peptide libraries were constructedin an M13 vector using techniques known to those skilled in the art.Adey et al. (1996) “Construction of Random Peptide Libraries inBacteriophage M13,” in Phage Display of Peptides and Proteins, Kay etal., eds., Academic Press, San Diego, Calif. Particularly, linear 8mers(L8), cyclic 6mers (C6) and single C (Cl) peptides were displayed asN-terminal extensions of the pIII bacteriophage protein. Thecharacteristics of the libraries are presented below in Table 3. TABLE3^(a) Number of Library Randomized Segment^(b,c) Sequences Linear 8mer AE X X X X X X X X G G 2.5 × 10¹⁰ (L8) (P)_(6 . . .) Cyclic 6mer A E C XX X X X X C 6.4 × 10⁷  (C6) (P)_(4 . . .) Single C (C1) A E X X X X X XX X G C 2.5 × 10¹⁰ (P)_(6 . . .)

[0176] Panning of the libraries was carried out using the techniquesdescribed by Smith et al. (1993) Methods in Enzymology 217:228, with theexception that the antibodies were absorbed directly to microtiterplates. 100 μl solutions containing representative monoclonal antibodies(1 Mg/ml of SEAM-2, SEAM-3, SEAM-5, SEAM-7, SEAM-12, SEAM-16, SEAM-18,and SEAM-28), or a corresponding concentration of control antibodies (amurine anti-MenB PS-specific monoclonal (2-1B), a human anti-Hib PSmonoclonal (ED8), and a murine monoclonal of irrelevant specificity(Laz2))were incubated overnight at 4° C. in microtiter plates (ImmunolonII). After washing the wells with PBS, Blocking Solution (5% (w/v)non-fat dry milk, 0.2% (w/v) Tween-20, 0.02% (w/v) sodium azide in PBS)was added to completely fill the wells, and the plates were thenincubated at ambient temperature for 3 hours. The blocked plates werewashed six times with PBS.

[0177] Approximately 10¹⁰ pfu of phage were added to triplicate wells ina total volume of 100 μl per well. The plates were incubated with thephage overnight at 40° C. Each well was then washed nine times with PBS,and the bound phage released by adding to each well 100 μl of 0.2 Mglycine, HCl (pH 2.2) buffer and incubating at ambient temperature for 1hour. The buffer solutions from respective triplicate wells werecombined, and the pH adjusted to 8 by addition of 20 μl 1.5 M Tris (pH8.8) buffer per 100 μl of solution. A freshly grown culture (2 ml) of E.coli (XL1-Blue) at a density of OD_(550nm)=0.4-0.6 in LB mediacontaining 0.2% (w/v) maltose and 12 μg/ml tetracycline (LB-mal, tetmedia) was added to the combined solutions of released phage. The cellsand phage were incubated at 37° C. for 20 minutes, after which 20 ml ofmedia was added. The cells were grown overnight at 37° C., then pelletedby centrifugation (5000×g for 10 minutes). The supernatant was filteredthrough a 0.2 μm membrane, and the phage precipitated by adding 0.15volumes of 20% (w/v) polyethylene glycol 8000, 4 M NaCl., and allowingthe mixture to stand at 4° C. overnight. Precipitated phage werecollected by centrifugation (10,000×g for 10 minutes), and thenresuspended in 20 ml PBS (approximately 10¹² pfu/ml).

[0178] Each panning was repeated 3 or 4 times for each screen. Finally,phage released from the final pan were used to infect XL1-Blue cells andseveral serial dilutions were plated directly on LB-agar plates.Individual plaques were selected and amplified in 5 ml cultures ofXL1-Blue (LB-mal, tet media). DNA from the phage was prepared usingQIA8-Prep™ columns (Quiagen) and sequenced using a Sequenase™ kit(Amersham) according to the manufacturer's instructions.

[0179] A total of 67 unique peptide sequences (Peptides Pep 1 - Pep 67)were selected by the SEAM monoclonal antibodies. These peptide sequencesare depicted in FIG. 7 as SEQ ID NOs. 1-67. Of these sequences, 13 wereidentified on more than occasion (Table 4). With one exception, none ofthe sequences selected by the control antibodies (2-1B, ED8 and Laz2)were identical or significantly homologous to those selected by the SEAMmonoclonal antibodies. The single exception (SEQ ID NO. 9) was selectedby both SEAM-3 and the Laz2 control antibody. However, this result waspossibly due to a cross-contamination between reagents since bothexperiments were conducted at the same time. TABLE 4 Number of IdenticalAntibody Peptide Sequence Isolates SEAM-2  Pep 10 3 (SEQ ID NO. 10)SEAM-2  Pep 13 2 (SEQ ID NO. 13) SEAM-2  Pep 14 2 (SEQ ID NO. 14)SEAM-3, 16, 18 Pep 1  37  (SEQ ID NO. 1) SEAM-5  Pep 2  3 (SEQ ID NO. 2)SEAM-7  Pep 3  5 (SEQ ID NO. 3) SEAM-7, 18 Pep 4  2 (SEQ ID NO. 4)SEAM-7, 18 Pep 5  2 (SEQ ID NO. 8) SEAM-12 Pep 6  4 (SEQ ID NO. 6)SEAM-18 Pep 7  3 (SEQ ID NO. 7) SEAM-18 Pep 12 4 (SEQ ID NO. 12) SEAM-28Pep 8  2 (SEQ ID NO. 8) SEAM-28 Pep 67 2 (SEQ ID NO. 67)

EXAMPLE 7 Characterization of the Peptide Mimetics

[0180] For characterization of the antibody binding to syntheticpeptides, the partially purified monoclonal antibodies were purifiedfurther on a BIOCAD® perfusion chromatography workstation using a PorosG/M protein G column (4.6mm×100 mm) with a column volume of 1.7 ml(PerSeptive Biosystems, Framingham, Mass.). The protein G column wasequilibrated with 10 column volumes of PBS buffer. Monoclonal antibodypreparations (2 ml) from either ascites or tissue culture resuspended inPBS were injected onto the protein G column. After washing with 5 columnvolumes of PBS buffer, monoclonal antibody was eluted from protein Gcolumn with a 0.2 M Glycine-HCl, 150 mM sodium chloride (pH 2.5) buffer.The eluted antibodies were monitored with internally equippedspectrophotometric detectors at both 220 nm and 280 nm, and the elutionpeak collected and stored at 4° C. The pH of each 1 ml fraction wasraised to 8.0 by adding 100 μl of 1.5 M Tris (pH 8.8) immediately uponcollection. Concentrations of the purified monoclonal antibodies weredetermined with a spectrophotometer from absorbance at 280 nm using anextinction coefficient of 0.71 mg⁻¹ ml cm⁻¹.

[0181] An ELISA was used to determine the ability of anti-NPr-MenB PSantibodies to recognize synthetic peptides corresponding to selectedpeptide mimetic sequences identified in Table 4. Synthetic peptides werepurchased from Biosynthesis (Lewisville, Tex.). To facilitate absorptionto the ELISA plate, the peptides were modified by the addition at theamino terminus of a hydrophobic tail (Lauryl-GLY-GLY). Further, thepeptides were carboxyl-terminal amides. The synthetic peptides (1 mg)were resuspended in 100 μl of dimethyl sulfoxide (Sigma, St. Louis, Mo.)and an aliquot was then diluted further in 50 mM Hepes (FisherScientific, Pittsburgh, Pa.) pH 8.0, 150 mM NaCl (Sigma, St. Louis, Mo.)and 0.02% sodium azide (Sigma, St. Louis, Mo.) to a peptideconcentration of 10 μg/ml. Microtiter plates (Immulon 24; DynatechLaboratories Inc., Chantilly, Va.) containing 100 μl/well of a 10 μg/mlpeptide solution in 50 mM Hepes buffer were incubated overnight at 4° C.After washing the plates 3 times with phosphate buffered saline (PBS, pH7.4), the wells were filled with 200 μl of Blocking Buffer and incubatedfor 1-2 hours at room temperature to block non-specific binding sites.The plates were then washed 5 times with Washing Buffer.

[0182] Various dilutions of the SEAM monoclonal antibodies (50 μl) to betested for peptide binding were added to duplicate plates containingeither 50 μl of Diluting Buffer or 50 μl of Diluting Buffer containing50 μg of soluble NPr-MenB PS per ml (final inhibitor concentration of 25μg/ml). The plates were then covered and incubated overnight at 4° C.The following day plates were washed 5 times with Washing Buffer, andthen incubated for 3 hours at 4° C. with 100 μl/well of alkalinephosphatase-conjugated anti-mouse polyclonal antibody, IgA +IgG +IgM(Zymed, South San Francisco, Calif.) diluted 1:2000 in Diluting Buffer.The plates were then washed 5 times with Washing Buffer, and 100 μl offreshly prepared substrate (p-nitrophenyl phosphate, Sigma, St. Louis,Mo.) diluted to 1 mg/ml in Substrate Buffer was added to each well.Absorbance values were measured after 30 minutes at 405 nm.

[0183] Representative binding data to the tethered Pep 4 and Pep 8 areshown in FIG. 8-A and 8-B, respectively. Several of the SEAManti-NPr-MenB PS monoclonal antibodies recognize these two peptides. Incontrast, irrelevant mouse monoclonal antibodies of the same isotypesshow no binding in this assay (data not shown). For some of the SEAManti-NPr-MenB PS monoclonal antibodies, the addition of NPr-MenB PS at25 μg/ml completely inhibited binding of the antibody to the peptides(e.g., SEAM-3). For other antibodies, there is either partial inhibitionof binding (e.g., SEAM-16 and SEAM-18), or no inhibition (SEAM-5). Assummarized in Table 5, there is a close correspondence between theconcentration-dependent binding of the SEAM anti-NPr-MenB PS monoclonalantibodies to NPr-MenB PS and the respective binding to particularsynthetic peptides. See, for example, the relative binding of antibodiesSEAM-3, SEAM-5, SEAM-7, SEAM-16, and SEAM-18 to NPr-MenB PS and to Pep8. TABLE 5 Relative Binding of SEAM Monoclonal Antibodies To NPr- ToSynthetic Lauryl-GLY-GLY-Peptides^(b) MenB PS^(a) Pep 1^(c) Pep 2 Pep 3Pep 4 Pep 6 Pep 7 Pep 8 Pep 9 SEAM-3 0.004 — — 0.016 0.014 — — 0.0090.019 SEAM-5 5 — 47 3 3 — — 3 23 SEAM-7 15 81 80 11 6 25 — 11 60 SEAM-0.08 0.2 — — 0.2 — — 0.06 — 16 SEAM- 0.14 0.8 — 0.8 0.4  1 — 0.2 — 18

EXAMPLE 8 Preparation of Peptide Mimetic Vaccine Compositions

[0184] Vaccine compositions containing synthetic peptides correspondingto the above-described peptide mimetic sequences were prepared asfollows.

[0185] Preparation of OMP Vesicles. OMP vesicles were prepared from thecapsular-deficient mutant strain of Neisseria meningitidis Group B(Strain M7), using a combination of the techniques described by Lowellet al. (1988) J. Expt. Med. 167:658-663 and Zollinger et al. (1979) J.Clin. Invest. 63:836-848. In brief, Neisseria meningitidis strain M7 (anoncapsular mutant strain derived from NmB), from an overnight cultureon chocolate agar plates incubated at 37° C., was used to inoculate two500 ml flasks of sterile Frantz medium (10.3 g of Na₂HPO₄, 10 g ofcasamino acids (Difco, Detroit, Mich.), 0.36 g of KCl, 0.012 f ofcysteine-HCl (Sigma, St. Louis, Mo.), and 25 ml of 40% glucose-40 mMMgSO₄ (Sigma, St. Louis, Mo.) in 1 L of water, pH 7.4). The bacteriawere grown from an initial OD of 0.1-0.2 to log phase (OD of 0.75-0.85)on a shaker at 180 rpm for 6-8 hours. The bacteria were inactivated with0.5% phenol solution for one hour at room temperature. The cells wereharvested by centrifuging for 30 minutes at 3000×g. The supernatant wasdecanted, and the cells were washed twice with PBS. The resultant pelletwas stored at −20° C.

[0186] The bacteria were then resuspended in 15 ml buffer containing0.05 M Tris-HCl, 0.15 M NaCl and 0.01M EDTA (pH 7.4), and then warmed to56° C. for 30 minutes. After cooling to room temperature, the suspensionwas sheared in a Polytron (Kinematica GmbH., Luzern, Switzerland) atfull speed for 3 minutes and then centrifuged at 16000×g for 15 minutes.The resulting pellet was resuspended with 10 ml buffer (500 mM sodiumchloride, 50 mM sodium phosphate), and treated with 5 ml of DetergentSolution (10% sodium deoxycholate (DOC) (Calbiochem, La Jolla, Calif.),0.15 M glycine (Biorad, Hercules, Calif.) and 30 mMethylenediaminetetraacetic acid (EDTA) (SIGMA, Saint Louis, Mo.). Thesuspension was centrifuged at 16,000×g for 15 minutes. The supernatantwas then collected and centrifuged at 100,000×g for 2 hrs. A pelletcontaining the outer membrane protein preparation was resuspended in 10ml of water and stored at 4° C.

[0187] The 10 ml suspension of outer membrane protein was retreated with5 ml of the Detergent Solution, and then warmed to 56° C. for 30minutes. After cooling, lipopolysaccharide (LPS) was removed from theouter membrane protein by chromatography, 2 ml at a time, using a 2cm×20 cm Sephadex G-100 column (Pharmacia Fine Chemicals, Piscataway,N.J.) in a second detergent solution (1% DOC, 0.05 M glycine, and 0.005M EDTA, pH 8.8). The peak fractions were collected, warmed to 30° C. andsterile-filtered through a 0.2 μm membrane filter directly into 4volumes of cold, filter-sterilized ethanol. This mixture was incubatedat 4° C. overnight. The resulting precipitate was collected bycentrifugation at 16,000×g for 10 minutes, and resuspended in 1 ml ofsterile distilled water. The resulting OMP preparation was soluble butslightly opalescent, and was stored at −60° C.

[0188] Preparation of Peptide/OMP Vesicles. Vaccines were prepared frompeptides Pep 5 and Pep 8, or from a mixture of peptides Pep 1- Pep 9. Tofacilitate hydrophobic complexing of the peptides to the OMP vesicle,each peptide was modified by the addition at the amino terminus of ahydrophobic tail (Lauryl-GLY-GLY) and a carboxyl amide as describedabove for the ELISA. For each vaccine, 5 mg of peptide was dissolved in100 μl dimethylsulfoxide (DMSO) (SIGMA, Saint Louis, Mo.). The resultingsolution was diluted to 750 μl in buffer containing 50 mM4-(-2-hydroxyethyl)-1-piperazineethanesulfonic Acid (Hepes), pH 8.0, and1 M potassium ferricyanide (SIGMA, Saint Louis, Mo.). 7.5 μg ofzwitterionic detergent (Empigen, Calbiochem, La Jolla, Calif.) was thenadded to the above peptide solution. After incubation at roomtemperature for 1 hour, each of the peptide solutions was combined with250 μl of outer membrane protein (OMP) vesicles (20 mg/ml) for a totalvolume of 1 ml. The solution was heated to 75° C. for 20 minutes. Aftercooling to room temperature, the OMP/Peptide mixture was added to aSlide-A-Lyzer (Pierce, Rockford, Ill.) with a 10,000 molecular weightcut off, and dialyzed in 1 L PBS overnight. The PBS solution (1 L) waschanged twice over 8 hours.

EXAMPLE 9 Immunization with OMP-Peptide Mimetic Vaccine Compositions

[0189] In order to assess the OMP-peptide vaccine compositions preparedin Example 8 above, the following study was carried out.

[0190] Animals: Balb/c and CD1 mice (Jackson Laboratory, Bar Harbor,Me.) were used for the immunogenicity studies. Mice were kept inquarantine for 2 weeks.

[0191] Vaccine Preparations: For the first injection, vaccine solutions(2 mg/ml total peptide/protein in PBS) were combined with equal volumesof complete Freund's adjuvant. (Sigma, St. Louis, Mo.) to yield a finalconcentration of 1 mg/ml of peptide/protein. For the subsequentinjections, similar vaccine compositions were prepared using incompleteFreunds adjuvant. The respective compositions were forced back and forththrough 2 syringes in order to obtain homogenous emulsions which werethen used in the immunizations.

[0192] Immunizations: Each treatment group included 4 Balb/c mice and 4CD1 mice. There were also control groups of 4 Balb/c and 4 CD1 mice thatwere not immunized. Individual treatment groups received doses of 5 μgor 50 μg of peptide, and 5 μg or 50 μg of OMP Vesicles, respectively.The vaccine composition was administered intraperitonealy (IP), in atotal volume of 5 or 50 μl, respectively. Immunizations were repeated at3 week intervals for a total of 3 immunizations. The animals were bledfrom the tail vein 1 and 4 weeks after the third immunization.

[0193] CD1 and Balb/c mice immunized with peptide Pep 8 complexed withOMP vesicles develop high anti-Pep 8 antibody responses as measured byELISA in serum obtained 4 weeks post-third immunization. Representativedata for the responses of the CD1 mice are shown in FIG. 9. Antibodybinding to tethered Pep 8 is inhibited by soluble Pep 8 (Acetyl-[Pep8]-Amide) but not by a soluble irrelevant peptide “R1”(Acetyl-GLN-TRP-GLU-ARG-THR-TYR-Amide (SEQ ID NO. 68)). Anti-Pep 8antibodies also were elicited in mice immunized with a combination ofnine peptides (peptides Pep 1-Pep 9/OMP), but not in mice immunized withPep S/OMP alone. This demonstrates the Pep 8-specific antibodies wereelicited by Pep 8-containing immunogens.

[0194]FIG. 10 summarizes the cross-reactivity of the CD1 mouse immunesera with NPr-MenB PS or NAc-MenB PS in an ELISA assay. All threeimmunogens (Pep 5/OMP, Pep 8/OMP, and peptides Pep 1-Pep 9/OMP) appearedto elicit serum antibodies cross-reactive with NPr-MenB PS, which werenot detected in the serum pool from the unimmunized control mice.However, the specificity of this antibody binding could not be confirmedsince there was no significant inhibition observed in wells containingsoluble NPr-MenB PS (data not shown). The ability of soluble Pep 8(Acetyl-[Pep 8]-Amide) to inhibit binding of the anti-Pep 8 serum poolsto the solid phase NPr-MenB PS also could not be verified since thepresence of this peptide resulted in significant increase in antibodybinding which was not detected in the presence of a soluble irrelevantpeptide “R1” (Acetyl-GLN-TRP-GLU-ARG-THR-TYR-Amide (SEQ ID NO. 68)).

[0195] Data from characterization of the extensive collection of SEAMmonoclonal antibodies indicate that the ability of an antibody to bindto NAc-MenB PS in an ELISA correlates with the presence of autoantibodyactivity as assessed by binding to PSA expressed by CHP-134neuroblastoma cells (see Table 1). FIG. 11 summarizes thecross-reactivity of the CD1 mouse immune sera with NAc-MenB PS in anELISA. None of the serum pooled from the peptide-vaccinated mice werepositive in this assay. In contrast, a SEAM anti-NPr-MenB PS monoclonalantibody with known autoantibody activity was strongly positive in thisassay when tested at 2.0 μg/ml. The lack of cross-reactivity of theanti-Pep antisera with NAc-MenB PS by ELISA indicates that theseantibodies do not have PSA-specific autoantibody activity.

[0196] Complement-mediated bactericidal activity of pooled CD1 sera frommice immunized with either 5 μg or 50 μg of Pep 8/OMP vaccine is shownin FIGS. 12A and 12B, respectively. At both doses, the Pep 8-containingvaccine elicited serum antibodies that were able to mediatebacteriolysis of MenB strain 8047 in the presence of human complement. Aportion of this antibody may have been elicited by the OMP vesicles usedas an adjuvant. However, at serum dilutions of 1:1000, 50% or. greaterof the bactericidal activity was mediated by the anti-Pep 8 antibodiesas demonstrated by inhibition of the reaction with Lauryl-GLY-GLY-Pep 5at a final serum concentration of 100 μg/ml.

[0197] Thus, novel MenB PS antibodies, molecular pepride mimeticscapable of eliciting bactericidal MenB antibody, and method forobtaining and using the same are diaclosed. Although preferredembodiments of the subject invention have been described in some detail,it is understood that obvious variations can be made without departingfrom the spirit and the scope of the invention as defined by theappended claims.

Deposits of Strains Useful in Practicing the Invention

[0198] Deposits of biologically pure cultures of the following hybridomacell lines were made with the American Type Culture Collection (ATCC)12301 Parklawn Drive, Rockville, Mass. The accession numbers indicatedwere assigned after successful viability testing and the requiste feeswere paid. The deposits were made under the provisions of the BudapestTreaty on the international Regonition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and the Regulations thereunder(Budapest Treaty). This assures maintenance of viable cultures for aperiod of thirty (30) years from the date of deposit. The organisms willbe made available by the ATCC under the terms of the Budapest Treaty andsubject to an agreement between Chiron decoration and the ATCC, whichassures permanent and unrestricted availability of the progeny to onedetermined by the U.S. Commissioner of Patents and Trademarks to beentitled thereto according to 35 U.S.C. §122 and the Comissioner's rulespursuant thereto (includes 17 C.F.R. §1.12 with particular reference to886 OG 638). Upon the granting of a patent, all restrictions on theavailability to the public of the deposited cultures will be irrevocablyremoved.

[0199] These deposits are provided merely as convenience to those ofskill in the art, and are not an admission that a deposit is requiredunder 35 U.S.C. §112. The nucleic acid sequences of these hybridomas, aswell as the amino acid sequences of the antibody molecules encodedthereby, are incorporated herein by reference and are controlling in theevent of any conflict with the description herein. A license may berequired to make, use, or sell the deposited materials, and no suchlicense is hereby granted. HYBRIDOMA Deposit Date ATCC No. SEAM-3 Aug.16, 1996 HB-12170 SEAM-18 Aug. 16, 1996 HB-12169 SEAM-2 July 30, 1997CRL-12380 SEAM-12 July 30, 1997 CRL-12381

[0200]

1 68 1 9 PRT Artificial Sequence Description of Artificial Sequencesequence from a phage display peptide library 1 Pro Leu Arg Ser Leu ArgSer Tyr Trp 1 5 2 10 PRT Artificial Sequence Description of ArtificialSequence sequence from a phage display peptide library 2 Ser Asn Cys GluIle Trp Arg Val Gly Cys 1 5 10 3 8 PRT Artificial Sequence Descriptionof Artificial Sequence sequence from a phage display peptide library 3Cys Met Arg Tyr Glu Ala Thr Cys 1 5 4 8 PRT Artificial SequenceDescription of Artificial Sequence sequence from a phage display peptidelibrary 4 Cys Gly Leu Pro Arg Phe Arg Cys 1 5 5 8 PRT ArtificialSequence Description of Artificial Sequence sequence from a phagedisplay peptide library 5 Tyr Cys Gln Ile Gln Gly Ser Cys 1 5 6 10 PRTArtificial Sequence Description of Artificial Sequence sequence from aphage display peptide library 6 Gln Val Pro Cys Ser Ser Arg Arg Gly Cys1 5 10 7 10 PRT Artificial Sequence Description of Artificial Sequencesequence from a phage display peptide library 7 Arg Tyr Gly Cys Leu LeuMet Arg Gly Cys 1 5 10 8 9 PRT Artificial Sequence Description ofArtificial Sequence sequence from a phage display peptide library 8 PheHis Cys Lys Val Asn Arg Gly Cys 1 5 9 10 PRT Artificial SequenceDescription of Artificial Sequence sequence from a phage display peptidelibrary 9 Ser Cys Arg Ser Lys Asn Ser Ala Gly Cys 1 5 10 10 8 PRTArtificial Sequence Description of Artificial Sequence sequence from aphage display peptide library 10 Thr Val Glu Thr Val Glu Ser Cys 1 5 118 PRT Artificial Sequence Description of Artificial Sequence sequencefrom a phage display peptide library 11 Tyr Gln Gly Pro Leu Gly Trp Arg1 5 12 8 PRT Artificial Sequence Description of Artificial Sequencesequence from a phage display peptide library 12 Cys Trp Pro Thr Leu GluGly Cys 1 5 13 8 PRT Artificial Sequence Description of ArtificialSequence sequence from a phage display peptide library 13 Cys Leu ThrSer Trp Ser Ser Cys 1 5 14 8 PRT Artificial Sequence Description ofArtificial Sequence sequence from a phage display peptide library 14 CysGly Leu Glu Leu Gln Gly Cys 1 5 15 8 PRT Artificial Sequence Descriptionof Artificial Sequence sequence from a phage display peptide library 15Cys Thr Thr Ile Met Cys Ser Thr 1 5 16 8 PRT Artificial SequenceDescription of Artificial Sequence sequence from a phage display peptidelibrary 16 Gly Tyr Glu Val Gln Pro Phe His 1 5 17 8 PRT ArtificialSequence Description of Artificial Sequence sequence from a phagedisplay peptide library 17 Val Ala Lys Thr Val Arg Pro Pro 1 5 18 8 PRTArtificial Sequence Description of Artificial Sequence sequence from aphage display peptide library 18 Trp Ala Ser Trp Val Gly Gly Pro 1 5 198 PRT Artificial Sequence Description of Artificial Sequence sequencefrom a phage display peptide library 19 Asp Asp Gly Tyr Glu Ile Arg Trp1 5 20 7 PRT Artificial Sequence Description of Artificial Sequencesequence from a phage display peptide library 20 Ser Arg Met Gly Gly ArgArg 1 5 21 8 PRT Artificial Sequence Description of Artificial Sequencesequence from a phage display peptide library 21 His Asn Lys Ser Lys LeuGlu Ala 1 5 22 8 PRT Artificial Sequence Description of ArtificialSequence sequence from a phage display peptide library 22 Gly His GlyAla Tyr Thr Arg Leu 1 5 23 8 PRT Artificial Sequence Description ofArtificial Sequence sequence from a phage display peptide library 23 LysSer Leu Asn Ala Met Val Leu 1 5 24 8 PRT Artificial Sequence Descriptionof Artificial Sequence sequence from a phage display peptide library 24Pro Trp Ser Arg Leu Lys Ser Pro 1 5 25 8 PRT Artificial SequenceDescription of Artificial Sequence sequence from a phage display peptidelibrary 25 Pro Ser Lys Gly Lys Val Leu Ser 1 5 26 8 PRT ArtificialSequence Description of Artificial Sequence sequence from a phagedisplay peptide library 26 Gly Pro Met Ser Ile Asp Leu Arg 1 5 27 8 PRTArtificial Sequence Description of Artificial Sequence sequence from aphage display peptide library 27 Arg Thr Glu Leu Gly Trp Arg Tyr 1 5 288 PRT Artificial Sequence Description of Artificial Sequence sequencefrom a phage display peptide library 28 Ser Asp Ser Gly Cys Tyr Gly Tyr1 5 29 8 PRT Artificial Sequence Description of Artificial Sequencesequence from a phage display peptide library 29 Cys Gly Thr Gln His ValGly Cys 1 5 30 8 PRT Artificial Sequence Description of ArtificialSequence sequence from a phage display peptide library 30 Cys Gly ThrHis Asp Leu Ala Cys 1 5 31 8 PRT Artificial Sequence Description ofArtificial Sequence sequence from a phage display peptide library 31 CysGln Lys Gly Ala Arg Gly Cys 1 5 32 8 PRT Artificial Sequence Descriptionof Artificial Sequence sequence from a phage display peptide library 32Cys Ser Arg Tyr Asn Gly Gly Cys 1 5 33 8 PRT Artificial SequenceDescription of Artificial Sequence sequence from a phage display peptidelibrary 33 Cys Gly Arg Ser Thr Glu Leu Cys 1 5 34 8 PRT ArtificialSequence Description of Artificial Sequence sequence from a phagedisplay peptide library 34 Cys Arg Asn Ser Gln Gly Tyr Cys 1 5 35 8 PRTArtificial Sequence Description of Artificial Sequence sequence from aphage display peptide library 35 Leu Asp Ser Gln Leu Arg Arg Thr 1 5 368 PRT Artificial Sequence Description of Artificial Sequence sequencefrom a phage display peptide library 36 Gly Trp Leu Phe Arg Gly Leu Met1 5 37 8 PRT Artificial Sequence Description of Artificial Sequencesequence from a phage display peptide library 37 Leu Asn Phe Lys Val ArgHis Asn 1 5 38 8 PRT Artificial Sequence Description of ArtificialSequence sequence from a phage display peptide library 38 Ala Lys SerVal His Tyr Gly Ile 1 5 39 8 PRT Artificial Sequence Description ofArtificial Sequence sequence from a phage display peptide library 39 CysVal Ala Leu Met Gly Gly Cys 1 5 40 10 PRT Artificial SequenceDescription of Artificial Sequence sequence from a phage display peptidelibrary 40 Cys Gln Lys Gly Ala Arg Ala Arg Gly Cys 1 5 10 41 8 PRTArtificial Sequence Description of Artificial Sequence sequence from aphage display peptide library 41 Phe Ala Ala Ala Leu Gly Gln Asn 1 5 428 PRT Artificial Sequence Description of Artificial Sequence sequencefrom a phage display peptide library 42 Tyr Ser His Trp Lys Trp Arg Trp1 5 43 8 PRT Artificial Sequence Description of Artificial Sequencesequence from a phage display peptide library 43 Gln Met Arg Pro Ala LeuAsn Ser 1 5 44 8 PRT Artificial Sequence Description of ArtificialSequence sequence from a phage display peptide library 44 Trp Leu AspArg Gly Ser Thr Pro 1 5 45 8 PRT Artificial Sequence Description ofArtificial Sequence sequence from a phage display peptide library 45 AspTrp Asp Arg Ala Val Val Leu 1 5 46 8 PRT Artificial Sequence Descriptionof Artificial Sequence sequence from a phage display peptide library 46Phe Pro Leu Leu Arg Gly Ala Arg 1 5 47 10 PRT Artificial SequenceDescription of Artificial Sequence sequence from a phage display peptidelibrary 47 Phe Ala Trp Ser Cys Thr Trp Pro Gly Cys 1 5 10 48 8 PRTArtificial Sequence Description of Artificial Sequence sequence from aphage display peptide library 48 Lys Leu His Val Gly Pro Arg Asn 1 5 498 PRT Artificial Sequence Description of Artificial Sequence sequencefrom a phage display peptide library 49 Leu Phe Pro Lys Pro Arg Leu Pro1 5 50 9 PRT Artificial Sequence Description of Artificial Sequencesequence from a phage display peptide library 50 Tyr Leu Gly Thr Ser ArgAsn Gly Leu 1 5 51 8 PRT Artificial Sequence Description of ArtificialSequence sequence from a phage display peptide library 51 Cys Gly ThrHis Asp Leu Ala Cys 1 5 52 9 PRT Artificial Sequence Description ofArtificial Sequence sequence from a phage display peptide library 52 CysGly Ser Ala Phe Ser Ala His Pro 1 5 53 10 PRT Artificial SequenceDescription of Artificial Sequence sequence from a phage display peptidelibrary 53 Ser Trp Trp His Asn Tyr Cys Pro Gly Cys 1 5 10 54 10 PRTArtificial Sequence Description of Artificial Sequence sequence from aphage display peptide library 54 Glu Arg Cys Ala Cys Gly Arg Gly Gly Cys1 5 10 55 10 PRT Artificial Sequence Description of Artificial Sequencesequence from a phage display peptide library 55 Glu Thr Lys Glu Arg GlyGlu Ser Gly Cys 1 5 10 56 11 PRT Artificial Sequence Description ofArtificial Sequence sequence from a phage display peptide library 56 AlaPhe Cys Cys Gly Ser Gly Thr Arg Gly Cys 1 5 10 57 10 PRT ArtificialSequence Description of Artificial Sequence sequence from a phagedisplay peptide library 57 Ala Phe Cys Gly Ser Gly Thr Arg Gly Cys 1 510 58 10 PRT Artificial Sequence Description of Artificial Sequencesequence from a phage display peptide library 58 Asn Leu Ser Ser Pro CysGly Arg Gly Cys 1 5 10 59 10 PRT Artificial Sequence Description ofArtificial Sequence sequence from a phage display peptide library 59 ValAla Cys Arg Ser Gly Met Gly Gly Cys 1 5 10 60 10 PRT Artificial SequenceDescription of Artificial Sequence sequence from a phage display peptidelibrary 60 Ile Arg Ser Gly Cys Arg Pro Val Gly Cys 1 5 10 61 9 PRTArtificial Sequence Description of Artificial Sequence sequence from aphage display peptide library 61 Cys Trp Lys Pro Gly Arg Ser Gly Cys 1 562 9 PRT Artificial Sequence Description of Artificial Sequence sequencefrom a phage display peptide library 62 Phe Val Arg Gly Val Gly Val GlyCys 1 5 63 10 PRT Artificial Sequence Description of Artificial Sequencesequence from a phage display peptide library 63 Gly Cys Trp Arg Trp IleGln Pro Gly Cys 1 5 10 64 10 PRT Artificial Sequence Description ofArtificial Sequence sequence from a phage display peptide library 64 PheAla Trp Ser Cys Thr Trp Pro Gly Cys 1 5 10 65 10 PRT Artificial SequenceDescription of Artificial Sequence sequence from a phage display peptidelibrary 65 Arg Cys Arg Gly His Gly Gly Pro Gly Cys 1 5 10 66 10 PRTArtificial Sequence Description of Artificial Sequence sequence from aphage display peptide library 66 Phe Ala Trp Ser Cys Thr Trp Pro Gly Cys1 5 10 67 10 PRT Artificial Sequence Description of Artificial Sequencesequence from a phage display peptide library 67 Cys Asn Leu Arg Met SerSer Ala Gly Cys 1 5 10 68 6 PRT Artificial Sequence Description ofArtificial Sequence soluble irrelevant peptide 68 Gln Trp Glu Arg ThrTyr 1 5

We claim:
 1. An isolated antibody directed against a Neisseriameningitidis serogroup B capsular polysaccharide derivative, whereinsaid antibody is not autoreactive.
 2. The antibody of claim 1 whereinsaid antibody does not cross-react with Neisseria meningitidis serogroupB capsular polysaccharide (MenB PS) in an ELISA.
 3. The antibody ofclaim 1 wherein said antibody displays functional activity against aNeisseria meningitidis serogroup B organism.
 4. The antibody of claim 1wherein said antibody is a monoclonal antibody.
 5. A unique Neisseriameningitidis serogroup B epitope capable of being bound by the antibodyof claim
 1. 6. A unique Neisseria meningitidis serogroup B epitopecapable of being bound by the antibody of claim
 2. 7. A unique Neisseriameningitidis serogroup B epitope capable of being bound by the antibodyof claim
 3. 8. A unique Neisseria meningitidis serogroup B epitopecapable of being bound by the antibody of claim
 4. 9. A hybridoma thatproduces the monoclonal antibody of claim
 4. 10. The hybridoma of claim9 having the identifying characteristics of a hybridoma cell lineselected from the group consisting of SEAM-2 (ATCC No. CRL-12380),SEAM-3 (ATCC No. HB-12170), SEAM-12 (ATCC No. HB-12169), and SEAM-18(ATCC No. CRL-12381).
 11. A method for isolating a molecular mimetic ofa unique epitope of Neisseria meningitidis serogroup B (MenB), saidmethod comprising: (a) providing a population of molecules comprising aputative molecular mimetic of a unique epitope of MenB; (b) contactingsaid population of molecules with the antibody of claim 1 underconditions that allow immunological binding between said antibody andsaid molecular mimetic, if present, to provide a complex; and (c)separating the complexes from non-bound molecules.
 12. The method ofclaim 11 wherein said population of molecules comprises a peptoidlibrary.
 13. The method of claim 11 wherein said population of moleculescomprises a peptide library.
 14. The method of claim 11 wherein saidpopulation of molecules comprises a phage-display library.
 15. Amolecular mimetic of a unique epitope of Neisseria meningitidisserogroup B (MenB), wherein said mimetic is isolated using the method ofclaim
 11. 16. A molecular mimetic of a unique epitope of Neisseriameningitidis serogroup B (MenB), wherein said mimetic is comprised of ananti-idiotypic antibody molecule produced using the antibody molecule ofclaim
 1. 17. A molecular mimetic of a unique epitope of Neisseriameningitidis serogroup B (MenB), wherein said mimetic is comprised of apeptide having an amino acid sequence that is substantially homologousto a sequence selected from the group consisting of SEQ ID NOs. 1-66,and SEQ ID NO.
 67. 18. The mimetic of claim 17, wherein said mimetic iscomprised of a peptide having an amino acid sequence that issubstantially homologous to SEQ ID NO.
 8. 19. A vaccine compositioncomprising a unique epitope of Neisseria meningitidis serogroup B (MenB)in combination with a pharmaceutically acceptable excipient.
 20. Avaccine composition comprising a molecular mimetic of a unique epitopeof Neisseria meningitidis serogroup B (MenB) in combination with apharmaceutically acceptable excipient.
 21. The vaccine composition ofclaim 20, wherein the molecular mimetic comprises an anti-idiotypicantibody molecule.
 22. The vaccine composition of claim 20, wherein themolecular mimetic comprises a nucleic acid molecule.
 23. The vaccinecomposition of claim 20, wherein the molecular mimetic comprises apeptide molecule.
 24. The vaccine composition of claim 23, wherein thepeptide molecule has an amino acid sequence that is substantiallyhomologous to a sequence selected from the group consisting of SEQ IDNOs. 1-66, and SEQ ID NO.
 67. 25. The vaccine composition of claim 19,wherein said epitope is covalently bound to a carrier molecule.
 26. Thevaccine composition of claim 20, wherein said molecular mimetic iscovalently bound to a carrier molecule.
 27. The vaccine composition ofclaim 23, wherein said peptide molecule is covalently bound to a carriermolecule.
 28. The vaccine composition of claim 19 further comprising anadjuvant.
 29. The vaccine composition of claim 20 further comprising anadjuvant.
 30. A method for preventing Neisseria meningitidis serogroup Band/or E. coli Kl disease in a mammalian subject, said method comprisingadministering an effective amount of the vaccine of claim 19 to saidsubject.
 31. A method for preventing Neisseria meningitidis serogroup Band/or E. coli Kl disease in a mammalian subject, said method comprisingadministering an effective amount of the vaccine of claim 20 to saidsubject.
 32. A method for preventing Neisseria meningitidis serogroup Band/or E. coli Kl disease in a mammalian subject, said method comprisingadministering an effective amount of the vaccine of claim 23 to saidsubject.
 33. A pharmaceutical composition comprising an antibodyaccording to claim 1 in combination with a pharmaceutically acceptablevehicle.
 34. A method for treating or preventing Neisseria meningitidisserogroup B and/or E. coli Kl disease in a mammalian subject, saidmethod comprising administering an effective amount of thepharmaceutical composition of claim 33 to said subject.